Manipulated acid alpha-glucosidase variant

Abstract

Pompe disease is an autosomal recessive lysosomal storage disorder caused by mutations in the gene encoding acid alpha-glucosidase (GAA). This disclosure provides engineered acid alpha-glucosidase polypeptides, recombinant polynucleotides encoding engineered acid alpha-glucosidase polypeptides, and methods for using engineered acid alpha-glucosidase polypeptides and recombinant polynucleotides for therapeutic purposes. This disclosure provides acid alpha-glucosidase polypeptides engineered to have improved properties, in particular, compared to naturally occurring human acid alpha-glucosidase.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 511,347, filed Jun. 30, 2023, which is hereby incorporated by reference in its entirety.

[0002] Reference to a Sequence Listing, Table, or Computer Program The sequence listing, created on Jun. 27, 2024, having a file size of 2,391,488 bytes and filed herewith under the name CX7 - 223WO1_ST26.xml, is part of this specification and is hereby incorporated by reference.

[0003] The present disclosure relates to engineered acid alpha - glucosidase (GAA) polypeptides, compositions thereof, polynucleotides encoding engineered acid alpha - glucosidase polypeptides, and the use of engineered polypeptides for therapeutic and other purposes.

Background Art

[0004] Pompe disease is an autosomal recessive lysosomal storage disorder caused by mutations in the gene encoding acid alpha-glucosidase (GAA). This genetic defect leads to a decrease or absence of acid alpha-glucosidase in body tissues. The resulting accumulation of glycogen in lysosomes causes lysosomal swelling and rupture, which can lead to cell damage, organelle dysfunction, and defects in other cells. There are two main forms of Pompe disease, including the classic infantile form and the late-onset form (childhood or adulthood), with some patients exhibiting an intermediate phenotype. Disease severity is related to the amount of enzyme activity present in the cells of the affected individual. The infantile form is the most severe and rapidly progressing form, typically having less than 1% acid alpha-glucosidase activity and causing significant glycogen accumulation in skeletal muscle, as well as the heart and other tissues (see, e.g., Hahn and Schanzer, Ann. Transl. Med., 2019, 7:283). In these patients, there is multisystem storage of accumulated lysosomes and non-lysosomal-bound glycogen in the heart, skeletal muscle, and brain tissue (see, e.g., Schoser, Ann. Transl. Med., 2019, 7:292). Patients have elevated creatine kinase levels, hypertrophic cardiomyopathy, stunted growth, hypotonia, and axial weakness. If left untreated, patients typically die within the first year of life due to cardiopulmonary failure. Survival beyond 18 months is exceptional.

[0005] The infantile form is distinguished from non-classical Pompe disease or late infantile Pompe disease, in which patients present with much less severe cardiac hypertrophy. Patients with late-onset Pompe disease typically experience progressive limb myopathy and respiratory failure. These patients present with prominent, though not exclusive, muscle lesions. Patients eventually require a wheelchair and / or mechanical ventilation. Respiratory failure is the leading cause of death in these patients. Some patients can synthesize a non-functional form of acid alpha-glucosidase, while others do not produce any cross-reactive immunomaterial of the natural enzyme. The human gene encoding acid alpha-glucosidase is localized to chromosome 17q25.2–q25.3, has been cloned, and sequenced (see, e.g., Peruzzo et al., Ann. Transl. Med., 2019, 7:278–287, and Martiniuk et al., DNA Cell. Biol., 1991, 10:283–292). Although numerous mutations have been reported within the gene, the pathological mechanisms that produce the wide range of phenotypes observed in affected patients remain unclear. Despite the availability of enzyme replacement therapy (ERT) using recombinant human acid alpha-glucosidase, better treatment and management options for affected patients are still needed. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Hahn and Schanzer,Ann.Transl.Med.,2019,7:283 [Patent Document 2] Schoser,Ann.Transl.Med.,2019,7:292 [Patent Document 3] Peruzzo et al.,Ann.Transl.Med.,2019,7:278-287 and Martiniuk et al.,DNA Cell.Biol.,1991,10:283-292 [Overview of the Initiative]

[0007] This disclosure provides acid alpha-glucosidase polypeptides that have been engineered to have improved properties, particularly compared to naturally occurring human acid alpha-glucosidases.

[0008] In some embodiments, the Disclosure provides an engineered acid alpha-glucosidase polypeptide or a bioactive fragment thereof, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity with respect to a reference sequence corresponding to residues 20-944 of even-numbered sequence codes 2, 12, and 14-754, wherein the amino acid sequence comprises one or more substitutions with respect to a reference sequence corresponding to residues 20-944 of sequence code 12 or 2, or to a reference sequence corresponding to sequence code 12 or 2.

[0009] In some embodiments, the manipulated acid alpha-glucosidase, or a biologically active fragment thereof, comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity with respect to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12 or 2, or to a reference sequence corresponding to SEQ ID NO: 12 or 2, wherein the amino acid sequence comprises one or more substitutions with respect to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12 or 2, or to a reference sequence corresponding to SEQ ID NO: 12 or 2.

[0010] In some embodiments, the manipulated acid alpha-glucosidase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity with respect to a reference sequence corresponding to residues 20-944 of even-numbered sequence numbers 14-754, or with respect to an even-numbered sequence number 14-754, wherein the amino acid sequence comprises one or more substitutions with respect to a reference sequence corresponding to residues 20-944 of sequence number 12 or 2, or with respect to a reference sequence corresponding to sequence number 12 or 2.

[0011] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least substitutions at amino acid positions 24, 28, 29, 39, 50, 62, 78, 87, 135, 150, 266, 267, 305, 437, 486, 522, 569, 670, 692, 711, 736, 750, 812, 830, 842, 871, 883, 894, 913, or 932, or combinations thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12, or to a reference sequence corresponding to SEQ ID NO: 12.

[0012] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase is at least one substitution: 24A / C / D / E / F / G / H / I / K / L / M / N / P / R / S / T / V / Y, 28A / C / D / E / F / G / H / K / L / P / Q / R / T / V / W, 29A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y, 39A / E / F / G / I / L / N / T, 50A / C / D / E / F / G / H / I / K / L / M / N / Q / R / S / T / W / Y, 62A / D / E / F / G / H / I / K / M / N / P / Q / S / T / V / Y, 78A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / V / W / Y, 87A / D / G / H / I / K / L / MN / Q / R / S / T / V / W, 135A / C / D / E / F / G / H / I / K / L / N / P / R / Y, 150T , 266A / D / E / H / K / Q / T, 267H / L / R / T / V, 305V, 437A / H / S, 486A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 522A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y, 569A / C / D / E / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y, 670A / D / E / F / G / H / I / K / L / M / N / Q / R / S / V / Y, 692A / C / D / E / F / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 7 11A / C / D / E / F / G / I / K / L / M / N / Q / R / S / T / V / W / Y, 736F / L, 750A / E / K / L / Q / R, 812A / D / G / S, 830D / E / F / G / H / L / M / N / Q / S / T / V / W / Y, 842A / C / D / E / F / G / H / K / L / M / N / Q / R / T / W, 871A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 883A / F / Q, 894A / C / D / E / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 913E / F / H / I / K / M / N / Q / S / W, or 932C / D / E / G / H / K / L / M / N / P / Q / R / S / T / W / Y, or combinations thereof, the amino acid positions are relative to the reference sequence corresponding to residues 20-944 of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0013] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase is at least one substitution: 24A / C / D / F / G / H / I / K / M / N / P / S / T / V / Y, 28A / C / D / E / F / G / H / K / Q / T / V / W, 29A / C / D / E / F / G / H / I / K / M / N / P / R / W / Y, 39A / E / F / G / I / L / N / T, 50A / C / D / E / F / H / I / K / M / N / R / S / T / W / Y, 62D / H / I / K / M / N / P / Q / Y, 78A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 87A / G / H / I / K / L / MN / Q / R / S / T / V / W, 135C / D / E / F / G / H / I / K / L / N / R / Y, 266A / D / E / H / K / Q, 267H / L / T / V, 305V, 437A / H, 486C / D / F / G / H / I / K / L / M / N / Q / R / S / V / W / Y, 522A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y, 569A / C / D / E / G / K / M / N / P / R / W, 670A / D / G / H / K / M / Y, 692A / D / E / H / K / L / M / N / T / W, 711D / E / I / K / M / N / Q / S / T / V / Y, 736F / L, 750E / K / L / Q / R, 812A / D / G / S, 830D / E / F / G / H / L / M / N / S / T / W / Y, 842A / C / D / F / H / K / L / M / N / Q / R / T / The amino acid positions include W, 871A / C / D / F / H / I / M / N / Q / T / V / W / Y, 883A / F / Q, 894A / D / E / H / I / K / L / M / N / S / T / V / W / Y, 913F / I / K / M / N / S, or 932C / D / E / G / H / K / L / M / N / P / Q / R / W / Y, or combinations thereof, and the amino acid positions are relative to the reference sequence corresponding to residues 20-944 of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0014] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes a sequence containing residues 20-944 of even-numbered sequence numbers 14-754, or a sequence containing even-numbered sequence numbers 14-754.

[0015] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes a sequence containing residues 20-944 of SEQ ID NOs: 14, 114, 126, 170, 250, 252, 394, 472, 488, or 506, or a sequence containing SEQ ID NOs: 14, 114, 126, 170, 250, 252, 394, 472, 488, or 506.

[0016] In some embodiments, the engineered acid alpha-glucosidase exhibits at least one improved property compared to a reference acid alpha-glucosidase. In some embodiments, the engineered acid alpha-glucosidase exhibits at least one improved property, selected from any combination of i), ii), iii), iv), v), vi), vii), viiii), and ix), compared to a reference acid alpha-glucosidase having a sequence corresponding to residues 20-944 of SEQ ID NO: 2 or 12, or a sequence corresponding to SEQ ID NO: 2 or 12, i) improved catalytic activity, ii) increased tolerance to pH 7, iii) increased tolerance to pH 4.4, iv) increased stability in lysosomes, v) increased intracellular expression, vi) increased intracellular uptake, vii) increased enzyme activity in cell lysates, viiii) increased stability in plasma / serum, and ix) decreased immunogenicity.

[0017] In a further embodiment, the Disclosure provides recombinant polynucleotides comprising polynucleotide sequences encoding the manipulated acid alpha-glucosidase disclosed herein.

[0018] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity with respect to a reference polynucleotide sequence corresponding to odd-numbered sequence numbers 13–753, or to a reference polynucleotide sequence corresponding to odd-numbered sequence numbers 13–753, wherein the polynucleotide encodes an acid alpha-glucosidase.

[0019] In some embodiments, the recombinant polynucleotide encoding the engineered acid alpha-glucosidase comprises a codon-optimized polynucleotide sequence for the expression of the engineered acid alpha-glucosidase.

[0020] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence containing nucleotide residues 58 to 2832 of odd-numbered sequence numbers 13 to 753, or a polynucleotide sequence containing odd-numbered sequence numbers 13 to 753.

[0021] In further embodiments, the disclosure provides an expression vector comprising a recombinant polynucleotide encoding the engineered acid alpha-glucosidase disclosed herein. In some embodiments, the expression vector comprises a control sequence operably ligated to the recombinant polynucleotide encoding the engineered acid alpha-glucosidase. In some embodiments, the control sequence is a promoter, for example, a heterologous promoter.

[0022] In another aspect, provided herein are host cells comprising an expression vector comprising the recombinant polynucleotide described herein. In some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the host cell is a mammalian cell, particularly a human cell. In some embodiments, the human cell is derived from a patient having a deficiency in acid alpha-glucosidase activity, for example, a patient suffering from Pompe disease.

[0023] In another aspect, host cells are used to produce the engineered acid alpha-glucosidase disclosed herein. In some embodiments, a method of producing an engineered acid alpha-glucosidase, the method comprising culturing a host cell comprising an expression vector under suitable conditions such that the engineered acid alpha-glucosidase is produced.

[0024] In another aspect, the present disclosure provides a pharmaceutical composition comprising an engineered acid alpha-glucosidase or a recombinant polynucleotide encoding an engineered acid alpha-glucosidase, comprising an expression vector comprising the recombinant polynucleotide. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0025] In another aspect, an engineered acid alpha-glucosidase or a recombinant polynucleotide encoding an engineered acid alpha-glucosidase is used to treat a subject having a deficiency in acid alpha-glucosidase activity. In some embodiments, a method for treating and / or preventing symptoms of acid alpha-glucosidase deficiency in a subject comprises administering to the subject in need thereof an effective amount of the engineered acid alpha-glucosidase disclosed herein or a recombinant polynucleotide encoding an engineered acid alpha-glucosidase.

[0026] In some embodiments, a pharmaceutical composition comprising engineered acid alpha-glucosidase or a recombinant polynucleotide encoding engineered acid alpha-glucosidase is administered to a subject.

[0027] In some embodiments, the subjects for treatment are those suffering from Pompe disease. In some embodiments, the subjects are infants or children. In some embodiments, the subjects are adults or adolescents.

[0028] In further embodiments, the disclosure provides the use of engineered acid alpha-glucosidase or recombinant polynucleotides encoding engineered acid alpha-glucosidase, or pharmaceutical compositions thereof for treating acid alpha-glucosidase activity deficiencies. In some embodiments, the acid alpha-glucosidase deficiency is Pompe disease. [Brief explanation of the drawing]

[0029] [Figure 1] A graph is provided showing the duration of stability of six GAA variants in a neutral pH cell culture medium, as described in Example 6. [Figure 2] A graph is provided showing the melting temperatures of six GAA variants at lysosomal pH (4.4) and slightly basic pH (7.4), as described in Example 6. [Figure 3] A graph is provided showing the duration of stability of six GAA variants when challenged by plasma treatment, as described in Example 6. [Figure 4] This graph provides the 4-MU-GLU hydrolytic activity in lysates derived from cultured Pompe patient-derived fibroblasts, treated with seven purified GAA variants as described in Example 6 for durations of 1 hour (Panel 4A), 4 hours (Panel 4B), 24 hours (Panel 4C), or 96 hours (Panel 4D), after which the GAA material was washed away and incubated at 37°C for up to 96 hours post-treatment. The values ​​are expressed as RFU activity. [Figure 5] This graph provides the 4-MU-GLU hydrolytic activity in lysates derived from cultured C2C12 GAA knockout myoblasts, treated with seven purified GAA variants as described in Example 6 for durations of 1 hour (Panel 5A), 4 hours (Panel 5B), 24 hours (Panel 5C), or 96 hours (Panel 5D), after which the GAA material was washed away and incubated at 37°C for up to 96 hours post-treatment. The values ​​are expressed as RFU activity. [Figure 6] This graph shows the 4-MU-GLU hydrolytic activity in the supernatant of cultures of C2C12 GAA knockout myoblasts transfected with plasmid DNA of six GAA variants. [Figure 7] The graphs provide showing GAA activity (4-MU-GLU hydrolysis shown in panel 7A and glycogen hydrolysis shown in panel 7B) in normalized lysates of C2C12 GAA knockout myoblasts transfected with plasmid DNA of six GAA variants. [Figure 8] Panel 8A shows the total of all high-quality GAA-derived peptides observed by mass spectrometry from each GAA variant in the ex vivo MHC II-related peptide proteomics assay (MAPPs assay), while Panel 8B shows the peptide frequency across donors, plotted against the region of the GAA sequence to which the peptide was treated for each GAA variant. In the MAPPs assay, PBMCs from healthy donors were differentiated into dendritic cells (antigen-presenting cells) and incubated in the presence of GAA variants (antigens). Next, HLA-binding peptides were eluted from HLA-DR molecules and identified by mass spectrometry to provide information on peptide treatment and presentation in antigen-presenting cells. These results indicate that the GAA variants of SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 14 significantly reduced treatment and decreased peptide presentation frequency compared to WT GAA and SEQ ID NO: 2. [Modes for carrying out the invention]

[0030] This disclosure provides engineered acid alpha-glucosidase (GAA) polypeptides and compositions thereof. In some embodiments, engineered acid alpha-glucosidase polypeptides are engineered to exhibit improved properties, including enhanced catalytic activity and improved acid stability, while reducing sensitivity to proteolysis. This disclosure also provides methods for using engineered acid alpha-glucosidase polypeptides (including compositions thereof) for therapeutic and other purposes.

[0031] Abbreviations and definitions With reference to this disclosure, the technical and scientific terms used in this description have meanings that are generally understood by those skilled in the art unless otherwise specifically defined.

[0032] It should be understood that the invention described herein may vary depending on the context in which it is used by those skilled in the art, and is therefore not limited to the specific methodologies, protocols, and reagents described. Accordingly, the terms defined immediately below are more fully described by referring to this application as a whole.

[0033] Furthermore, the section headings provided herein should not be construed as limitations on the various aspects or embodiments of the invention that may be obtained by referring to this application as a whole.

[0034] As used herein, the singular terms “a,” “an,” and “the” also include plural references unless the context clearly indicates otherwise.

[0035] As used herein, the term “comprising” and its cognates are used in their comprehensive sense (i.e., equivalent to the term “including” and its corresponding cognates).

[0036] When the description of an embodiment uses the term “comprising” and its cognates, it should also be understood that the embodiment may also be described using the language “consisting essentially of” or “consisting of.”

[0037] Furthermore, a numerical range includes a numerical range of the numbers that define the range. Thus, all numerical ranges disclosed herein are intended to encompass all narrower numerical ranges that fall within such a wider numerical range, as if all narrower numerical ranges were expressly described herein. All maximum (or minimum) numerical limits disclosed herein are also intended to encompass all lower (or higher) numerical limits, as if all lower (or higher) numerical limits were expressly described herein.

[0038] "Approximately" means the allowable error for a particular value. In some cases, "approximately" means within 0.05%, 0.5%, 1.0%, or 2.0% of a given range of values. In some cases, "approximately" means within 1 standard deviation, 2 standard deviations, 3 standard deviations, or 4 standard deviations of a given value. In some cases, "approximately" encompasses values ​​within 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of a given value.

[0039] The "EC" number refers to the enzyme nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numbering system for enzymes based on the chemical reactions they catalyze.

[0040] "ATCC" refers to the American Type Culture Collection, a collection of biorepositories that includes genes and strains.

[0041] "NCBI" refers to the National Center for Biological Information and the sequence databases it provides.

[0042] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to polymers of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation).

[0043] "Amino acids" are referred herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. The abbreviations used for genetically coded amino acids are conventional and are as follows: Alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glycine (Gly or G), glutamine (Gln or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). When a three-letter abbreviation is used, unless it is specifically preceded by "L" or "D" or is clear from the context in which the abbreviation is used, an amino acid can be in either the L-configuration or the D-configuration with respect to the α-carbon (Cα). For example, "Ala" represents alanine without specifying the configuration with respect to the α-carbon, while "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively. When a single-letter abbreviation is used, uppercase letters represent amino acids in the L-configuration with respect to the α-carbon, and lowercase letters represent amino acids in the D-configuration with respect to the α-carbon. For example, "A" represents L-alanine and "a" represents D-alanine. When a polypeptide sequence is presented as a series of one- or three-letter abbreviations (or a mixture thereof), the sequence is presented in the direction from amino (N) to carboxyl (C), according to common convention.

[0044] "Fusion protein," "chimeric protein," and "chimera" refer to hybrid proteins created by the combination of two or more polynucleotides that originally encoded separate proteins. In some embodiments, fusion proteins are created by recombinant techniques (e.g., molecular biology techniques known in the art).

[0045] "Acid alpha-glucosidase," "acid α-glucosidase," "acid alpha-glucosidase polypeptide," "lysosomal alpha-glucosidase," and "GAA" refer to enzymes within a family of glycogen-degrading enzymes present in lysosomes (EC 3.2.1.20). This enzyme may also be called "alpha-1,4-glucosidase," "α-1,4-glucosidase," "acid maltase," "glucoinvertase," "glucosidosucrase," "lysosomal alpha-glucosidase," "lysosomal α-glucosidase," "maltase," or "maltase-glucoamylase." One reaction catalyzed by the enzyme is the hydrolysis of terminal non-reducing (1 to 4) linked alpha-D-glucose residues, which results in the release of alpha-D-glucose. As used herein, the term "rhGAA" refers to recombinant human alpha-glucosidase acid.

[0046] Pompe disease refers to type II glycogen storage disease, an autosomal recessive genetic disorder that typically causes metabolic disorders characterized by lysosomal storage of glycogen in skeletal muscle and other tissues. It is characterized based on the age of onset, organ involvement, severity, and rate of progression. A more severe form is infant-onset Pompe disease (IOPD), which develops in infants. Another form, called late-onset Pompe disease (LOPD), involves disease onset before 12 months of age but does not have IOPD-related cardiomyopathy, and occurs in all individuals with disease onset after 12 months of age. Synonyms for Pompe disease include acid alpha-glucosidase deficiency, acid maltase deficiency, GAA deficiency, glycogen storage disease type II, GSD II, GSD2, and glycogenosis type II.

[0047] "Polynucleotide," "nucleic acid," or "oligonucleotide" is used herein to refer to a polymer comprising at least two nucleotides, wherein the nucleotide is either a deoxyribonucleotide or a ribonucleotide, or a mixture of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations used to genetically encode nucleosides are conventional and include: adenosine (A), guanosine (G), cytidine (C), thymidine (T), and uridine (U). Unless otherwise specified, the abbreviated nucleoside may be either a ribonucleoside or a 2'-deoxyribonucleoside. Nucleosides may be designated as either a ribonucleoside or a 2'-deoxyribonucleoside, individually or based on aggregates. When a polynucleotide, nucleic acid, or oligonucleotide sequence is presented as a series of single-letter abbreviations, the sequence is presented in the 5' to 3' direction according to common convention, and phosphates are not indicated. The term "DNA" refers to deoxyribonucleic acid. The term "RNA" refers to ribonucleic acid. Polynucleotides or nucleic acids may be single-stranded or double-stranded, and may contain both single-stranded and double-stranded regions.

[0048] When used in reference to cells, polynucleotides, or polypeptides, “manipulated,” “recombinant,” “not naturally occurring,” and “variant” refer to materials that are otherwise not naturally occurring or identical thereto, but are produced or derived from synthetic materials and / or modified by manipulation using recombinant technology, or materials corresponding to the natural or native forms of those materials.

[0049] "Wild-type" and "naturally occurring" refer to forms found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in living organisms that can be isolated from a natural source and has not been intentionally modified by human manipulation. In some embodiments, "wild-type" and "naturally occurring" refer to forms found in nature that have normal function and / or activity.

[0050] A "coding sequence" refers to a portion of nucleic acid (for example, a gene) that codes for the amino acid sequence of a protein.

[0051] "Percent Sequence Identity (%)" is used herein to refer to a comparison across polynucleotides and polypeptides, determined by comparing two optimally aligned sequences across a comparison frame, where the portion of the polynucleotide or polypeptide sequence in the comparison frame may contain additions or deletions (i.e., gaps) compared to a reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions in which identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison frame, and multiplying the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions in which either of the identical nucleic acid bases or amino acid residues occurs in both sequences or in conjunction with the gaps to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison frame, and multiplying the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will understand that there are many established algorithms available for aligning two sequences. Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by the similarity method search of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), by computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection known in the art.Examples of suitable algorithms for determining sequence identity percentage and sequence similarity include, but are not limited to, the BLAST and BLAST 2.0 algorithms, which are described by Altschul et al. (see Altschul et al., J.Mol.Biol., 1990, 215:403-410 and Altschul et al., Nucleic Acids Res., 1997, 25(17):3389-3402, respectively). Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information website. This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in the database sequence, either match or satisfy a threshold score T of some positive value. T is referred to as the neighbor word score threshold (see Altschul et al., above). These initial neighbor word hits serve as seeds to initiate a search for longer HSPs containing them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for pairs of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. The extension of word hits in each direction stops when the cumulative alignment score has fallen by quantity X from its maximum achieved value, when the cumulative score becomes zero or less due to the accumulation of alignments of one or more negative-scoring residues, or when either sequence reaches its end. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, by default, a word length (W) of 11, an expected value (E) of 10, M=5, N=-4, and comparison of both strands.For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 score matrix by default (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary determinations of sequence alignment and % sequence identity can be performed using the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI) with the provided default parameters.

[0052] A “reference sequence” refers to a defined sequence used as the basis for sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotides or amino acid residues, at least 25 residues, at least 50 residues, or at least 100 residues in length or full length of the nucleic acid or polypeptide. Since each of two polynucleotides or polypeptides may (1) contain sequences similar between the two sequences (i.e., parts of the complete sequence) and (2) further contain sequences that differ between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides across a “frame of comparison” to identify and compare local regions of sequence similarity. In some embodiments, the “reference sequence” may be based on a primary amino acid sequence, and the reference sequence may have one or more changes from the primary sequence.

[0053] A "comparison frame" refers to a conceptual segment of consecutive nucleotide positions or amino acid residues from which a sequence can be compared to a reference sequence. In some embodiments, a comparison frame is at least 15–20 consecutive nucleotides or amino acids, and the portion of the sequence within the comparison frame may contain no more than 20% additions or deletions (i.e., gaps) compared to the reference sequence (which is free of additions or deletions) for optimal alignment of the two sequences. In some embodiments, a comparison frame may be longer than 15–20 consecutive residues and may optionally include frames of 30, 40, 50, 100, or longer.

[0054] The terms "corresponding to," "referencing," or "against" refer to the numbering of residues in a particular reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the number or position of residues in a given polymer is specified in relation to the reference sequence, not to the actual numerical position of the residues in the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as an engineered acid alpha-glucosidase, can be aligned to a reference sequence by introducing gaps to optimize the residue matching between the two sequences. In these cases, although gaps exist, the numbering of residues in the given amino acid or polynucleotide sequence is done with respect to the reference sequence to which it is aligned.

[0055] A "mutation" refers to a change in a nucleic acid sequence. In some embodiments, a mutation results in a change to the encoded polypeptide sequence (i.e., compared to the original sequence without the mutation). In some embodiments, a mutation includes substitutions such that different amino acids are produced. In some alternative embodiments, a mutation includes additions such that amino acids are added to (e.g., insertions) the original polypeptide sequence. In some further embodiments, a mutation includes deletions such that amino acids are removed from the original polypeptide sequence. Any number of mutations can be present in a given sequence.

[0056] An "amino acid difference" or "residue difference" refers to the difference between an amino acid residue at a position in the polypeptide sequence and an amino acid residue at a corresponding position in the reference sequence. The position of the amino acid difference is generally referred to herein as "Xn," where n is the corresponding position in the reference sequence, and the residue difference is based on this. For example, "residue difference at position X24 compared to SEQ ID NO: 12" refers to the difference in amino acid residues at the polypeptide position corresponding to position 24 in SEQ ID NO: 12. Therefore, if the reference polypeptide of SEQ ID NO: 12 has tryptophan at position 24, "residue difference at position X24 compared to SEQ ID NO: 2" is an amino acid substitution of any residue other than tryptophan at the polypeptide position corresponding to position 24 in SEQ ID NO: 12. In most cases herein, a difference in a particular amino acid residue at a given position is indicated as "XnY," where "Xn" identifies the corresponding position as described above, and "Y" is a single-letter identifier of the amino acid found in the manipulated polypeptide (i.e., a residue different from that in the reference polypeptide). In some cases (for example, as shown in Table 3-1), the Disclosure also provides a specific amino acid difference represented by the conventional notation “AnB,” where A is a single-letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is a single-letter identifier of the residue substitution in the sequence of the manipulated polypeptide. In some embodiments, the amino acid difference, e.g., substitution, is represented by the abbreviation “nB” without the identifier of the residue in the reference sequence. In some embodiments, the phrase “amino acid residue nB” indicates the presence of an amino acid residue in the manipulated polypeptide, which may or may not be a substitution in the context of the reference sequence.

[0057] In some cases, the polypeptides of this disclosure may contain one or more amino acid residue differences from a reference sequence, which are indicated by a list of specific positions where residue differences from the reference sequence exist. In some embodiments, if more than one amino acid can be used at a particular residue position of the polypeptide, the various amino acid residues that can be used are separated by " / " (e.g., X24A / X24C or X24A / C or 24A / C). In some embodiments, an amino acid residue is selected from a variety of alternative amino acid residues listed for that residue position. In some embodiments, the polypeptide variant contains more than one substitution. These substitutions are separated by slashes (e.g., L24W / L28S or 24W / 28S) or semicolons for readability, as described below. In some cases, as stated above, "X" does not precede the position number in this application.

[0058] "Amino acid substitution set" and "substitution set" refer to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, a substitution set includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. In some embodiments, a substitution set refers to a set of amino acid substitutions present in any of the acid alpha-glucosidase polypeptides listed in any of the Tables of Examples. In these substitution sets, individual substitutions are separated by a semicolon (e.g., L24W;L28S) or a slash (" / "; e.g., L24W / L28S or 24W / 28S). In some embodiments, "substitution" includes amino acid deletions and can be represented by the "-" symbol.

[0059] "Conservative amino acid substitution" refers to the substitution of a residue at a different residue having a similar side chain, and therefore typically involves the substitution of an amino acid in a polypeptide at an amino acid within the same or similar defined class of amino acids. As an example, but not limited to, an amino acid having an aliphatic side chain may be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid having a hydroxyl side chain may be substituted with another amino acid having a hydroxyl side chain (e.g., serine and threonine); an amino acid having an aromatic side chain may be substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid having a basic side chain may be substituted with another amino acid having a basic side chain (e.g., lysine and arginine); an amino acid having an acidic side chain may be substituted with another amino acid having an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid may be substituted with another hydrophobic or hydrophilic amino acid, respectively.

[0060] A “non-conservative substitution” refers to the substitution of an amino acid in a polypeptide by an amino acid with significantly different side-chain properties. Non-conservative substitutions can use amino acids between, rather than within, defined groups and affect (a) the structure of the peptide backbone in the region of substitution (e.g., proline in glycine), (b) charge or hydrophobicity, or (c) the bulk of the side chain. Examples of non-conservative substitutions, though not limited to them, may include acidic amino acids substituted with basic or aliphatic amino acids, aromatic amino acids substituted with small amino acids, and hydrophilic amino acids substituted with hydrophobic amino acids.

[0061] "Deletion" refers to a modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions may include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids constituting the reference polypeptide, while retaining activity and / or improved properties of the modified polypeptide. Deletions may be directed to the internal and / or terminal portions of the polypeptide. In various embodiments, deletions may include continuous segments or be discontinuous.

[0062] "Insertion" refers to a modification of a polypeptide by adding one or more amino acids from a reference polypeptide. Insertions can be located in the interior of the polypeptide, or at the carboxyl or amino terminus. Insertions as used herein include fusion proteins known in the art. Insertions can be a continuous segment of amino acids, or they can be separated by one or more amino acids in a naturally occurring polypeptide.

[0063] As used interchangeably herein, “functional fragment” or “biologically active fragment” refers to a polypeptide having an amino-terminal and / or carboxy-terminal deletion(s) and / or internal deletion, but whose remaining amino acid sequence is identical to the corresponding position in the sequence being compared (e.g., the fully-length manipulated acid alpha-glucosidase of the present invention) and which retains substantially all of the activity of the full-length polypeptide.

[0064] "Isolated polypeptide" refers to a polypeptide substantially isolated from other contaminants naturally associated with it (e.g., proteins, lipids, and polynucleotides). This term encompasses polypeptides removed or purified from their naturally occurring environment or expression system (e.g., host cells or in vitro synthesis). The engineered acid alpha-glucosidase polypeptide may be present intracellularly, in cell culture medium, or prepared in various forms such as lysates or isolated preparations. Therefore, in some embodiments, the engineered acid alpha-glucosidase polypeptide can be an isolated polypeptide.

[0065] A "substantially pure polypeptide" refers to a composition in which the polypeptide species present is the dominant species (i.e., abundant in moles or by weight compared to any other individual polymer species in the composition), and is generally considered substantially purified if the species of interest constitutes at least about 50% of the polymer species present in moles or by weight. Generally, a substantially pure polypeptide composition contains about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all polymer species present in the composition in moles or by weight. In some embodiments, the species of interest is purified to the point of intrinsic homogeneity (i.e., the contaminating species cannot be detected in the composition by conventional detection methods), and the composition consists essentially of a single polymer species. Solvent species, low molecular weight (less than 500 Daltons), and elemental ion species are not considered polymer species. In some embodiments, the isolated polypeptide is a substantially pure polypeptide composition.

[0066] "Improved enzyme properties" refers to an engineered acid alpha-glucosidase polypeptide that exhibits improvements in any enzyme properties compared to a reference acid alpha-glucosidase polypeptide, which may be a wild-type acid alpha-glucosidase polypeptide or another engineered acid alpha-glucosidase polypeptide. Improved properties include, but are not limited to, increased protein expression, increased thermal activity, increased thermal stability, increased pH activity, increased stability, increased enzyme activity, increased specific activity, increased resistance to substrate or end-product inhibition, increased chemical stability, improved solvent stability, increased tolerance to acidic, neutral, or basic pH, increased tolerance to proteolytic activity (i.e., decreased sensitivity to proteolysis), reduced aggregation, increased solubility, decreased immunogenicity, improved post-translational modifications (e.g., glycosylation), altered temperature profile, and increased lysosomal stability.

[0067] "Increased enzyme activity" or "improved catalytic activity" refers to improved properties of an engineered acid alpha-glucosidase polypeptide, which can be expressed by an increase in specific activity (e.g., product produced / time / weight protein) or an increase in the percentage of substrate-to-product conversion (e.g., the percentage of substrate-to-product conversion within a given time using a given amount of acid alpha-glucosidase) compared to a reference acid alpha-glucosidase enzyme. Exemplary methods for determining enzyme activity are provided in the examples. m , V max or k cat Any properties related to enzyme activity, including the classic enzymatic properties, may be affected, and changes in these properties may result in increased enzyme activity. The improvement in enzyme activity can range from approximately 1.1 times the enzyme activity of the corresponding wild-type enzyme to 2, 5, 10, 20, 25, 50, 75, 100, 150, 200 times, or more than the naturally occurring acid alpha-glucosidase or another engineered acid alpha-glucosidase from which the acid alpha-glucosidase polypeptide is derived.

[0068] In some embodiments, acid alpha-glucosidase activity can be measured by any suitable method known in the art (e.g., standard assays such as monitoring changes in the spectrophotometric properties of reactants or products). In some embodiments, the amount of product produced can be measured by monitoring the fluorescence (excitation at 355 nm, emission at 460 nm) after hydrolysis of the 4-methylumbelliferyl-alpha-D-glucopyranoside (4-MUGlu) molecule, as provided in the examples. Comparison of enzyme activity is performed using defined preparations of the enzyme, defined assays under set conditions, and one or more defined substrates, as described in further detail herein. Generally, when comparing lysates, the number of cells and the amount of protein being assayed, as well as the use of identical expression systems and identical host cells, should be determined to minimize variations in the amount of enzyme produced by the host cells and present in the lysate.

[0069] "Improved tolerance to acidic pH" means that the manipulated acid alpha-glucosidase according to the present invention has increased stability (e.g., higher retention activity at approximately pH 4-4.8 after exposure to acidic pH for a specific period, e.g., 1 hour, up to 24 hours) compared to a reference acid alpha-glucosidase or another enzyme.

[0070] "Improved tolerance to neutral pH" means that the manipulated acid alpha-glucosidase according to the present invention has increased stability (e.g., higher retention activity at approximately pH 7 after exposure to neutral pH for a specific period, e.g., 1 hour, up to 24 hours) compared to a reference acid alpha-glucosidase or another enzyme.

[0071] "Improved cellular uptake" means that the engineered acid alpha-glucosidase provided herein exhibits increased endocytosis to cells compared to a reference acid alpha-glucosidase (including wild-type acid alpha-glucosidase) or another enzyme. In some embodiments, the cells are cultured Pompe patient-derived cells (higher retained intracellular activity after incubation with cells cultured over a specific period compared to a reference acid alpha-glucosidase or another enzyme). In some additional embodiments, the engineered acid alpha-glucosidase provided herein exhibits higher retained intracellular activity with cells cultured over a specific period compared to a reference acid alpha-glucosidase (including wild-type acid alpha-glucosidase) or another enzyme. In some additional embodiments, the period is about 4 hours, but in some other embodiments, the period is less than 4 hours (e.g., 1, 2, or 3 hours), and in some alternative embodiments, the period is greater than 4 hours (e.g., 5, 6, 7, 8, or more).

[0072] "Reduced immunogenicity" and "decreased immunogenicity" mean that the engineered acid alpha-glucosidases provided herein are expected to induce or have a reduced immune response compared to wild-type or another reference acid alpha-glucosidase.

[0073] As used herein, “physiological pH” means the pH range commonly found in the blood of the subject (e.g., human).

[0074] "Neutral pH" (used, for example, to refer to improved stability to basic pH conditions or increased tolerance to basic pH) means a pH of approximately 7.

[0075] "Basic pH" (used, for example, to refer to improved stability to basic pH conditions or increased tolerance to basic pH) means a pH greater than 7, for example, a pH range from 7 to 11.

[0076] "Acid pH" (used, for example, to refer to improved stability to acidic pH conditions or increased tolerance to acidic pH) means a pH below 7, e.g., a pH range of approximately 1.5 to 4.8.

[0077] "Conversion" refers to the enzymatic (or bioconversion) of a substrate(s) to its corresponding product(s). "Conversion percentage" refers to the percentage of a substrate converted to a product within a certain period of time under specific conditions. Therefore, the "enzymatic activity" or "activity" of an acid alpha-glucosidase polypeptide can be expressed as the "conversion percentage" from substrate to product over a specific period of time.

[0078] "Preferred reaction conditions" refers to the conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffer, cosolvent, etc.) on which the acid alpha-glucosidase polypeptide of this application can convert a substrate into a desired product compound. Exemplary "preferred reaction conditions" are provided in this application and illustrated by the examples. "Loading," e.g., "compound loading" or "enzyme loading," refers to the concentration or amount of components in the reaction mixture at the start of the reaction. "Substrate" in the context of an enzymatic conversion reaction process refers to the compound or molecule acted upon by the acid alpha-glucosidase polypeptide. "Product" in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the acid alpha-glucosidase polypeptide on the substrate.

[0079] "Codon-optimized" refers to the modification of codons in a protein-coding polynucleotide to those preferred for use in a particular organism, so that the encoded protein is more efficiently expressed in that organism. While the genetic code is degenerate in that most amino acids are represented by a few codons (called "synonymous" codons), codon usage by a particular organism is not random and is well known to be biased towards certain codon triplets. This bias in codon usage can be higher with respect to a given gene, a gene of common function or ancestral origin, a high-expression protein compared to a low-copy-number protein, and aggregated protein-coding regions of an organism's genome. In some embodiments, a polynucleotide encoding an acid alpha-glucosidase enzyme can be codon-optimized for optimal production from a host organism selected for expression.

[0080] In this specification, “control sequence” means all components necessary or advantageous for the expression of the polynucleotide and / or polypeptide of this application. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leaders, polyadenylated sequences, propeptide sequences, promoter sequences, signal peptide sequences, start sequences, and transcription termination factors. In some embodiments, the control sequence includes at least a promoter, as well as transcription and translation stop signals.

[0081] "Operably linked" or "operatively linked" is defined herein as a configuration in which the control sequence is appropriately positioned (i.e., functionally related) to a polynucleotide of interest and is appropriately encoded by the target polypeptide, so that the control sequence directs or modulates the expression of the polynucleotide.

[0082] A "promoter sequence" refers to a nucleic acid sequence recognized by a host cell for the expression of a target polynucleotide, such as a coding sequence. The promoter sequence contains transcriptional regulatory sequences that mediate the expression of the target polynucleotide. The promoter may be any nucleic acid sequence exhibiting transcriptional activity in a selected host cell, including mutant, cleaved, and hybrid promoters, and may be obtained from a gene encoding either homologous or heterologous extracellular or intracellular polypeptides in the host cell.

[0083] A “vector” refers to a polynucleotide construct for introducing a polynucleotide sequence into a cell. In some embodiments, the vector is an “expression vector” operably ligated to a polypeptide encoded by the polynucleotide, which may influence the expression of the polynucleotide of interest in a suitable host. In some embodiments, the expression vector has a promoter sequence operably ligated to the polynucleotide sequence (e.g., an introduced gene) to drive expression in a host cell, and in some embodiments, also includes a transcription termination factor sequence.

[0084] "Expression" includes, but is not limited to, transcription, post-transcriptional modification, translation, and post-translational modification, any steps involved in the production of polypeptides. In some embodiments, this term also encompasses the secretion of polypeptides from cells.

[0085] "Culturing" refers to the growth of a population of cells under any suitable conditions (e.g., using liquid, gel, or solid media).

[0086] "Produce" refers to the production or expression of proteins and / or other compounds by cells. This term is intended to encompass any steps involved in polypeptide production, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, this term also encompasses the secretion of polypeptides from cells.

[0087] "Heterogeneous" or "recombinant" refers to the relationship between two or more nucleic acid or protein sequences (e.g., promoter sequences, signal peptides, termination factor sequences) that originate from different sources and are not inherently associated.

[0088] "Host cell" and "host line" refer to a host suitable for expression vectors containing polynucleotides (e.g., polynucleotides encoding acid alpha-glucosidase variants) provided herein. In some embodiments, the host cell is a prokaryotic or eukaryotic cell transformed or transfected with a vector constructed using recombinant DNA technology, as is known in the art.

[0089] A "therapeutic drug" refers to a compound administered to a person exhibiting signs or symptoms of a disease that has a beneficial or desirable medical effect.

[0090] A “gene therapy vector” refers to a vehicle or carrier suitable for delivering polynucleotides to cells for therapeutic effects. In some embodiments, vectors include, but are not limited to, non-viral vectors such as adenoviruses (AV), adeno-associated viruses (AAV), lentiviruses (LV), and liposomes, which encapsulate a gene (e.g., a therapeutic gene) or polynucleotide sequence for delivery to cells or tissues. The invention is not intended to be limited to any particular gene therapy vector, as the use of any vehicle suitable for a given setting can be found. Gene therapy vectors may be designed to deliver genes to a specific species or host, or more general applicability may be found.

[0091] Gene therapy refers to the delivery of genes, polydeoxyribonucleotides, or polynucleotide sequences (or more) to cells or tissues using gene therapy vectors to modify those cells or tissues for the prevention or treatment of disease. Gene therapy may include replacing disease-causing mutant genes with healthy copies of the gene or functional variants of the gene, or inactivating or "knocking out" improperly functioning mutant genes. In some embodiments, gene therapy is used to treat diseases in patients.

[0092] "MRNA therapy" refers to the treatment or prevention of disease by delivering mRNA polyribonucleotide sequences to cells or tissues and modifying those cells or tissues. In some embodiments, the mRNA polynucleotide sequences for delivery to cells or tissues are formulated in liposomes, for example, but not limited to these. In some embodiments, mRNA therapy is used to treat a disease in a patient.

[0093] "Cell therapy" refers to the delivery of exogenously modified living cells to a patient to provide a deficient gene for treating or preventing a disease. The modified cells are then reintroduced into the body.

[0094] "Compositions" and "Formulations" encompass products comprising at least one operated acid alpha-glucosidase of the present disclosure, intended for any preferred use (e.g., pharmaceutical compositions, dietary / nutritional supplements, animal feed, etc.).

[0095] "Pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a mammalian subject (e.g., human), comprising a pharmaceutically effective amount of an engineered acid alpha-glucosidase polypeptide incorporated in the present invention and an acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises a recombinant polynucleotide encoding the engineered acid alpha-glucosidase, for example, in the form of a gene therapy vector.

[0096] "Pharmacologically acceptable" means a material that can be administered to a subject without causing undesirable biological effects or without harmful interaction with any of the components it contains that have the desired biological activity.

[0097] When used in reference to pharmaceutical compositions, "carrier" means any of the standard pharmaceutical carriers, buffers, and excipients, such as stabilizers, preservatives, and adjuvants.

[0098] "Excipients" refers to any pharmaceutically acceptable additives, carriers, diluents, adjuvants, or other components other than the active pharmaceutically active ingredient (API; e.g., a manipulated acid alpha-glucosidase polypeptide or a recombinant polynucleotide encoding acid alpha-glucosidase). Excipients are typically included for formulation and / or administration purposes.

[0099] "Administering" and "to administer" the composition mean providing the composition of the present invention to a subject (for example, a person affected by Pompe disease).

[0100] "Effective quantity" means the amount sufficient to produce the desired result. Those skilled in the art can determine the effective quantity by using routine experiments.

[0101] When used in relation to the symptoms of a disease / condition, “therapeutic effective dose” refers to the amount and / or concentration of a compound (e.g., a modified acid alpha-glucosidase polypeptide or a recombinant polynucleotide encoding a modified acid alpha-glucosidase) that alleviates, reduces or eliminates one or more symptoms of the disease / condition, or prevents or delays the onset of the symptom(s). When used in relation to a disease / condition, “therapeutic effective dose” refers to the amount and / or concentration of a composition (e.g., a modified GAA polypeptide or a recombinant polynucleotide encoding a modified acid alpha-glucosidase) that alleviates, reduces or eliminates the disease / condition. In some embodiments, the term is used to refer to the amount of a composition that elicits a biological (e.g., medical) response in a tissue, system, or animal subject as determined by a researcher, physician, veterinarian, or other clinician.

[0102] As used herein, “treating” or “treating” a disease, disorder, or syndrome includes (i) preventing the development of the disease, disorder, or syndrome in a subject, i.e., preventing the development of the clinical symptoms of the disease, disorder, or syndrome in animals that may be exposed to or predisposed to the disease, disorder, or syndrome but have not yet experienced or exhibited the symptoms of the disease, disorder, or syndrome; (ii) inhibiting the disease, disorder, or syndrome, i.e., halting its progression; and (iii) reducing the disease, disorder, or syndrome, i.e., causing regression of the disease, disorder, or syndrome. Thus, the terms “treating,” “treating,” and “treatment” encompass preventative (e.g., prophylactic) and palliative treatments. As is known in the art, adjustments to systemic and topical delivery, as well as adjustments with respect to age, weight, overall health, sex, diet, timing of administration, drug interactions, and severity of symptoms may be necessary, which can be confirmed by routine experiments by those skilled in the art.

[0103] The term "subjects" includes mammals such as humans, non-human primates, livestock, companion animals, and laboratory animals (e.g., rodents and lagomorphs). This term is intended to encompass both males and females.

[0104] "Patient" means any person who is being evaluated for a disease, being treated for a disease, or experiencing a disease.

[0105] "Infant" refers to a child from one month old to approximately one year old. As used herein, the term "neonatal" refers to a child up to 28 days old. The term "premature" refers to an infant born after completing 20 weeks of gestation but before the normal term, and generally weighing between approximately 500 and 2499 grams at birth. "Very low birth weight infant" is an infant weighing less than 1500g at birth.

[0106] "Child" refers to a person who has not reached the legal age to consent to a medical or research procedure. In some embodiments, this term refers to a person between birth and adolescence.

[0107] "Adult" refers to a person who has reached the legal age of the relevant jurisdiction (e.g., 18 years of age). In some embodiments, this term refers to any fully grown and mature organism. In some embodiments, the term "youth" refers to a person who is under 18 years of age but has reached sexual maturity.

[0108] Manipulated GAA polypeptide This disclosure provides engineered acid alpha-glucosidase polypeptides characterized by improved properties compared to wild-type acid alpha-glucosidase or a reference engineered acid alpha-glucosidase polypeptide. The engineered acid alpha-glucosidase polypeptides described herein are engineered to have, in particular, improved activity, plasma stability, cellular uptake, intralysosomal stability, and / or acid pH stability. Engineered acid alpha-glucosidase polypeptides have been found to be used in therapeutic applications, such as for the treatment of conditions associated with acid alpha-glucosidase deficiency.

[0109] In one embodiment, the Disclosure provides an engineered acid alpha-glucosidase polypeptide or a bioactive fragment thereof, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity with respect to a reference sequence corresponding to residues 20-944 of even-numbered sequence codes 2, 12, and 14-754, wherein the amino acid sequence comprises one or more substitutions with respect to a reference sequence corresponding to residues 20-944 of sequence code 12 or 2, or to a reference sequence corresponding to sequence code 12 or 2.

[0110] In some embodiments, the manipulated acid alpha-glucosidase, or a biologically active fragment thereof, comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity with respect to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12 or 2, or to a reference sequence corresponding to SEQ ID NO: 12 or 2, wherein the amino acid sequence comprises one or more substitutions with respect to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12 or 2, or to a reference sequence corresponding to SEQ ID NO: 12 or 2.

[0111] In some embodiments, the manipulated acid alpha-glucosidase, or a biologically active fragment thereof, comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity with respect to the reference sequence corresponding to residues 20-944 of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12, wherein the amino acid sequence comprises one or more substitutions with respect to the reference sequence corresponding to residues 20-944 of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0112] In some embodiments, the manipulated acid alpha-glucosidase, or a biologically active fragment thereof, comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity with respect to a reference sequence corresponding to residues 20-944 of even-numbered sequence numbers 14-754, or with respect to an even-numbered sequence number 14-754, wherein the amino acid sequence comprises one or more substitutions with respect to a reference sequence corresponding to residues 20-944 of sequence number 12 or 2, or with respect to a reference sequence corresponding to sequence number 12 or 2.

[0113] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least substitutions at amino acid positions 24, 28, 29, 39, 50, 62, 78, 87, 135, 150, 266, 267, 305, 437, 486, 522, 569, 670, 692, 711, 736, 750, 812, 830, 842, 871, 883, 894, 913, or 932, or combinations thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12 or 2, or to a reference sequence corresponding to SEQ ID NO: 12 or 2.

[0114] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase is at least substituted or has amino acid residues: 24A / C / D / E / F / G / H / I / K / L / M / N / P / R / S / T / V / Y, 28A / C / D / E / F / G / H / K / L / P / Q / R / T / V / W, 29A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y, 39A / E / F / G / I / L / N / T, 50A / C / D / E / F / G / H / I / K / L / M / N / Q / R / S / T / W / Y, 62A / D / E / F / G / H / I / K / M / N / P / Q / S / T / V / Y, 78A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / V / W / Y, 87A / D / G / H / I / K / L / MN / Q / R / S / T / V / W, 135A / C / D / E / F / G / H / I / K / L / N / P / R / Y, 150T , 266A / D / E / H / K / Q / T, 267H / L / R / T / V, 305V, 437A / H / S, 486A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 522A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y, 569A / C / D / E / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y, 670A / D / E / F / G / H / I / K / L / M / N / Q / R / S / V / Y, 692A / C / D / E / F / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 711A / C / D / E / F / G / I / K / L / M / N / Q / R / S / T / V / W / Y, 736F / L, 750A / E / K / L / Q / R, 812A / D / G / S, 830D / E / F / G / H / L / M / N / Q / S / T / V / W / Y, 842A / C / D / E / F / G / H / K / L / M / The amino acid positions include N / Q / R / T / W, 871A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 883A / F / Q, 894A / C / D / E / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 913E / F / H / I / K / M / N / Q / S / W, or 932C / D / E / G / H / K / L / M / N / P / Q / R / S / T / W / Y, or combinations thereof, where the amino acid positions are relative to the reference sequence corresponding to residues 20-944 of SEQ ID NO: 12 or 2, or to the reference sequence corresponding to SEQ ID NO: 12 or 2.

[0115] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase is at least substituted W24E / A / C / D / F / G / H / I / K / L / M / N / P / R / ST / V / Y, S28A / C / D / E / F / G / H / K / L / P / Q / R / T / V / W, T29A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y, Q39A / E / F / G / I / L / N / T, V50A / C / D / E / F / G / H / I / K / L / M / N / Q / R / S / T / W / Y, L62A / D / E / F / G / H / I / K / M / N / P / Q / S / T / V / Y, E78A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / V / W / Y, E87A / D / G / H / I / K / L / M / N / Q / R / S / T / V / W, Q135A / C / D / E / F / G / H / I / K / L / N / P / R / Y, S150T, N26 6A / D / E / H / K / Q / T, K267H / L / R / T / V, G437A / H / S, E486A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, V522A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y, T5 69A / C / D / E / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y, T670A / D / E / F / G / H / I / K / L / M / N / Q / R / S / V / Y, G692A / C / D / E / F / H / I / K / L / M / N / Q / R / S / T / V / W / Y, H711A / C / D / E / F / G / I / K / L / M / N / Q / R / S / T / V / W / Y, M736F / L, P750A / E / K / L / Q / R, E812A / D / G / S, K830D / E / F / G / H / L / M / N / Q / S / T / V / W / Y, S842A / C / D / E / F / G / H / K / L / M The amino acid positions include / N / Q / R / T / W, E871A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, H883A / F / Q, G894A / C / D / E / H / I / K / L / M / N / Q / R / S / T / V / W / Y, R913E / F / H / I / K / M / N / Q / S / W, A932C / D / E / G / H / K / L / M / N / P / Q / R / S / T / W / Y, or combinations thereof, where the amino acid positions are relative to the reference sequence corresponding to residues 20-944 of SEQ ID NO: 12 or 2, or to the reference sequence corresponding to SEQ ID NO: 12 or 2.

[0116] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase is at least substituted or has amino acid residues: 24A / C / D / F / G / H / I / K / M / N / P / S / T / V / Y, 28A / C / D / E / F / G / H / K / Q / T / V / W, 29A / C / D / E / F / G / H / I / K / M / N / P / R / W / Y, 39A / E / F / G / I / L / N / T, 50A / C / D / E / F / H / I / K / M / N / R / S / T / W / Y, 6 2D / H / I / K / M / N / P / Q / Y, 78A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 87A / G / H / I / K / L / MN / Q / R / S / T / V / W, 135C / D / E / F / G / H / I / K / L / N / R / Y, 266A / D / E / H / K / Q, 267H / L / T / V, 305V, 437A / H, 486C / D / F / G / H / I / K / L / M / N / Q / R / S / V / W / Y, 522A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y, 569A / C / D / E / G / K / M / N / P / R / W, 670A / D / G / H / K / M / Y, 692A / D / E / H / K / L / M / N / T / W, 711D / E / I / K / M / N / Q / S / T / V / Y, 736F / L, 750E / K / L / Q / R, 812A / D / G / S, 830D / E / F / G / H / L / M / N / S / T / W / Y, 842A / C / D / F / H / K / L / M / N / Q / R / The amino acid positions include T / W, 871A / C / D / F / H / I / M / N / Q / T / V / W / Y, 883A / F / Q, 894A / D / E / H / I / K / L / M / N / S / T / V / W / Y, 913F / I / K / M / N / S, or 932C / D / E / G / H / K / L / M / N / P / Q / R / W / Y, or combinations thereof, with the amino acid positions relative to the reference sequence corresponding to residues 20-944 of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0117] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase is at least substituted W24A / C / D / F / G / H / I / K / M / N / P / S / T / V / Y, S28A / C / D / E / F / G / H / K / Q / T / V / W, T29A / C / D / E / F / G / H / I / K / M / N / P / R / W / Y, Q39A / E / F / G / I / L / N / T, V50A / C / D / E / F / H / I / K / M / N / R / S / T / W / Y, L62D / H / I / K / M / N / P / Q / Y, E78A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, E87A / G / H / I / K / L / MN / Q / R / S / T / V / W, Q135C / D / E / F / G / H / I / K / L / N / R / Y, N266A / D / E / H / K / Q, K267H / L / T / V, L305V, G437A / H, E486C / D / F / G / H / I / K / L / M / N / Q / R / S / V / W / Y, V522A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y, T569A / C / D / E / G / K / M / N / P / R / W, T670A / D / G / H / K / M / Y, G692A / D / E / H / K / L / M / N / T / W, H711D / E / I / K / M / N / Q / S / T / V / Y, M736F / L, P750E / K / L / Q / R, E812A / D / G / S, K830D / E / F / G / H / L / M / N / S / T / W / Y, S842A / C / D / F / H / K / L / M / N / Q / R / The amino acid positions include T / W, E871A / C / D / F / H / I / M / N / Q / T / V / W / Y, H883A / F / Q, G894A / D / E / H / I / K / L / M / N / S / T / V / W / Y, R913F / I / K / M / N / S, or A932C / D / E / G / H / K / L / M / N / P / Q / R / W / Y, or combinations thereof, with the amino acid positions relative to the reference sequence corresponding to residues 20-944 of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0118] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 305. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 305V. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution L305V.

[0119] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 24. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 24A / C / D / F / G / H / I / K / M / N / P / S / T / V / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 24A / C / D / E / F / G / H / I / K / L / M / N / P / R / ST / V / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution W24A / C / D / F / G / H / I / K / M / N / P / S / T / V / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase contains at least the substitution W24E / A / C / D / F / G / H / I / K / L / M / N / P / R / ST / V / Y.

[0120] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 28. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 28A / C / D / E / F / G / H / K / Q / T / V / W. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 28A / C / D / E / F / G / H / K / L / P / Q / R / T / V / W. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution S28A / C / D / E / F / G / H / K / Q / T / V / W. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution S28A / C / D / E / F / G / H / K / L / P / Q / R / T / V / W.

[0121] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 29. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 29A / C / D / E / F / G / H / I / K / M / N / P / R / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 29A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution T29A / C / D / E / F / G / H / I / K / M / N / P / R / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase contains at least the substitution T29A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y.

[0122] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 39. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 39A / E / F / G / I / L / N / T. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution Q39A / E / F / G / I / L / N / T.

[0123] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 50. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 50A / C / D / E / F / H / I / K / M / N / R / S / T / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 50A / C / D / E / F / G / H / I / K / L / M / N / Q / R / S / T / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution V50A / C / D / E / F / H / I / K / M / N / R / S / T / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase contains at least the substitution V50A / C / D / E / F / G / H / I / K / L / M / N / Q / R / S / T / W / Y.

[0124] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 62. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 62D / H / I / K / M / N / P / Q / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 62A / D / E / F / G / H / I / K / M / N / P / Q / S / T / V / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution L62D / H / I / K / M / N / P / Q / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution L62A / D / E / F / G / H / I / K / M / N / P / Q / S / T / V / Y.

[0125] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 78. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 78A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 78A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / V / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution E78A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase contains at least the substitution E78A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / V / W / Y.

[0126] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 87. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 87A / G / H / I / K / L / MN / Q / R / S / T / V / W. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 87A / D / G / H / I / K / L / M / N / Q / R / S / T / V / W. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution E87A / G / H / I / K / L / MN / Q / R / S / T / V / W. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution E87A / D / G / H / I / K / L / M / N / Q / R / S / T / V / W.

[0127] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 135. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 135C / D / E / F / G / H / I / K / L / N / R / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 135A / C / D / E / F / G / H / I / K / L / N / P / R / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution Q135C / D / E / F / G / H / I / K / L / N / R / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution Q135A / C / D / E / F / G / H / I / K / L / N / P / R / Y.

[0128] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 150. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 150T. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution S150T.

[0129] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 266. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 266A / D / E / H / K / Q. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 266A / D / E / H / K / Q / T. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least the substitution N266A / D / E / H / K / Q. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least the substitution N266A / D / E / H / K / Q / T.

[0130] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 267. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 267H / L / T / V. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 267H / L / R / T / V. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least the substitution K267H / L / T / V. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least the substitution K267H / L / R / T / V.

[0131] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 437. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 437H. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 437A / H / S. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution G437H. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution G437A / H / S.

[0132] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 486. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 486C / D / F / G / H / I / K / L / M / N / Q / R / S / V / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 486A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution E486C / D / F / G / H / I / K / L / M / N / Q / R / S / V / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase contains at least the substitution E486A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y.

[0133] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 522. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 522A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 522A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution V522A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase contains at least the substitution V522A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y.

[0134] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 569. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 569A / C / D / E / G / K / M / N / P / R / W. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 569A / C / D / E / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution T569A / C / D / E / G / K / M / N / P / R / W. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase contains at least the substitution T569A / C / D / E / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y.

[0135] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 670. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 670A / D / G / H / K / M / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 670A / D / E / F / G / H / I / K / L / M / N / Q / R / S / V / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution T670A / D / G / H / K / M / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution T670A / D / E / F / G / H / I / K / L / M / N / Q / R / S / V / Y.

[0136] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 692. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 692A / D / E / H / K / L / M / N / T / W. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 692A / C / D / F / H / I / K / L / M / N / Q / R / S / T / V / W / Y / E. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least the substitution G692A / D / E / H / K / L / M / N / T / W. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least the substitution G692A / C / D / E / F / H / I / K / L / M / N / Q / R / S / T / V / W / Y.

[0137] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 711. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 711D / E / I / K / M / N / Q / S / T / V / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 711A / C / D / E / F / G / I / K / L / M / N / Q / R / S / T / V / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution H711D / E / I / K / M / N / Q / S / T / V / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase contains at least the substitution H711A / C / D / E / F / G / I / K / L / M / N / Q / R / S / T / V / W / Y.

[0138] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 736. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 736F / L. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution M736F / L.

[0139] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 750. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 750E / K / L / Q / R. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 750A / E / K / L / Q / R. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution P750E / K / L / Q / R. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution P750A / E / K / L / Q / R.

[0140] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 812. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 812A / D / G / S. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least the substitution E812A / D / G / S.

[0141] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 830. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 830D / E / F / G / H / L / M / N / S / T / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 830D / E / F / G / H / L / M / N / Q / S / T / V / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least the substitution K830D / E / F / G / H / L / M / N / S / T / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least the substitution K830D / E / F / G / H / L / M / N / Q / S / T / V / W / Y.

[0142] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 842. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 842A / C / D / F / H / K / L / M / N / Q / R / T / W. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 842A / C / D / E / F / G / H / K / L / M / N / Q / R / T / W. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution S842A / C / D / F / H / K / L / M / N / Q / R / T / W. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution S842A / C / D / E / F / G / H / K / L / M / N / Q / R / T / W.

[0143] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 871. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 871A / C / D / F / H / I / M / N / Q / T / V / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 871A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution E871A / C / D / F / H / I / M / N / Q / T / V / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase contains at least the substitution E871A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y.

[0144] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 883. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 883A / F / Q. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least the substitution H883A / F / Q.

[0145] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 894. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 894A / D / E / H / I / K / L / M / N / S / T / V / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 894A / C / D / E / H / I / K / L / M / N / Q / R / S / T / V / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution G894A / D / E / H / I / K / L / M / N / S / T / V / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase contains at least the substitution G894A / C / D / E / H / I / K / L / M / N / Q / R / S / T / V / W / Y.

[0146] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 913. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 913F / I / K / M / N / S. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 913E / F / H / I / K / M / N / Q / S / W. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least the substitution R913F / I / K / M / N / S. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least the substitution R913E / F / H / I / K / M / N / Q / S / W.

[0147] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution at amino acid position 932. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 932C / D / E / G / H / K / L / M / N / P / Q / R / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least a substitution or amino acid residue 932C / D / E / G / H / K / L / M / N / P / Q / R / S / T / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least the substitution A932C / D / E / G / H / K / L / M / N / P / Q / R / W / Y. In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase contains at least the substitution A932C / D / E / G / H / K / L / M / N / P / Q / R / S / T / W / Y.

[0148] In some embodiments, the manipulated acid alpha-glucosidase contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity with respect to the manipulated acid alpha-glucosidase having substitutions or sets of substitutions as shown in Table 3-1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12 or 2, or to a reference sequence corresponding to SEQ ID NO: 12 or 2.

[0149] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least substitutions at amino acid positions as shown in Table 3-1, where the amino acid positions are relative to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12 or 2, or to a reference sequence corresponding to SEQ ID NO: 12 or 2.

[0150] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least one substitution as shown in Table 3-1, where the amino acid position is relative to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12 or 2, or to a reference sequence corresponding to SEQ ID NO: 12 or 2.

[0151] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes at least the substitution set shown in Table 3-1, where the amino acid positions are relative to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2.

[0152] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase is as follows: amino acid positions 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 305 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 87 1 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 871 / 883 / 894 / 913 / 932, 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 78 / 87 / 1 35 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 4 37 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 670 / 692 / 71 1 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932,24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842S / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913, or 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 2 The sequence includes at least a substitution set at 67 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812S / 830 / 842 / 871 / 883 / 894 / 913 / 932, where the amino acid positions are relative to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, specific amino acid substitutions at positions in the substitution set are selected from the substitutions described herein for each amino acid position.

[0153] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase is at least the substitution set 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 305V / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78 E / 87E / 135P / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A , 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486Q / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 84 2S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842E / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78G / 87E / 135Q / 150S / 266N / 267K / 43 7G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894S / 913R / 932A,24W / 28S / 29A / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A / 830K / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62S / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 7 36M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932R、24W / 28A / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135 Q / 150S / 266N / 267K / 437G / 486E / 522V / 569A / 670T / 692G / 711H / 736M / 75 0P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 5 0V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569R / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842W / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29C / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267L / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87Q / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62D / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62H / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135R / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871A / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50S / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569G / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692R / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830N / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894R / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267T / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24E / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135G / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692V / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87R / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913Q / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711L / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87L / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87V / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 、 29T / 39Q / 50V / 62L / 78Y / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569C / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87W / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670A / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569Q / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62K / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894C / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569H / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711V / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522S / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24F / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569M / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711S / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24Y / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87G / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78I / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78R / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24C / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78W / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522F / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522P / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830V / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711M / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28T / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569K / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24S / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 48 6E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 88 3H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135L / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 7 8E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711 H / 736M / 750P / 812E / 830K / 842R / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 5 69T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842T / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 13 5Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894T / 913R / 932A, 24W / 28Q / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266H / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62P / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692、 G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39L / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486D / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711G / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24N / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711W / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871R / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522W / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894L / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883A / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522G / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522L / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670V / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62G / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 48 6E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 88 3H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692N / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 7 8E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569V / 670T / 692G / 711 H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 5 69T / 670I / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87A / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 13 5Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894A / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266A / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28R / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871G / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522I / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711Y / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50E / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62V / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87S / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62N / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87H / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486L / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913E / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711R / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39G / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24R / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266D / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39T / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 2、 8S / 29T / 39Q / 50C / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24A / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87K / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29E / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78L / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24P / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24D / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692K / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29D / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50Y / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486K / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28F / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569S / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883Q / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50F / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28W / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267V / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830E / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62E / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569I / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28V / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486V / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871V / 883H / 894G / 913R / 932A、24W / 28S / 29S / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28C / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830L / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28K / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871N / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50L / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932Q、24W / 28S / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711F / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569E / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78F / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522R / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711E / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V、 / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842F / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50N / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135D / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78S / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24V / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913S / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78M / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670Q / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62T / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 5 69T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894M / 913R / 932A, 24W / 28S / 29T / 39Q / 50K / 62L / 78E / 87E / 13 5Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 75 0P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 5 0V / 62L / 78N / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750L / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750R / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569D / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24I / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486N / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894V / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830D / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894W / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78V / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522A / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670M / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62Y / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750K / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78A / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 7 8E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711 H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894K / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437S / 486E / 522V / 5 69T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692E / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 13 5Q / 150S / 266N / 267K / 437G / 486E / 522V / 569P / 670T / 692G / 711H / 736M / 75 0P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 5 0V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569N / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135N / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522Y / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R、 / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736F / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932T、24G / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842K / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932W、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692Q / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135F / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871F / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670Y / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692L / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135E / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670F / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50H / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28P / 29T / 39Q / 50V / 62L / 7 8E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711 H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 5 69T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883F / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437H / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62I / 78E / 87E / 13 5Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 75 0P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 5 0V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830W / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692S / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29K / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670E / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486R / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135A / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39I / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87N / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87M / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135C / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932L、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670G / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830F / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522H / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28D / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522Q / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50A / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842 S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437、 G / 486E / 522V / 569T / 670S / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830M / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842A / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87T / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522T / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24K / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871K / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 48 6E / 522V / 569T / 670T / 692G / 711H / 736M / 750E / 812E / 830K / 842S / 871E / 88 3H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830T / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50I / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 5 69T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932E, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266Q / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39A / 50V / 62L / 78E / 87E / 13 5Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 75 0P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24T / 28S / 29T / 39Q / 5 0V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 48 6E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871T / 88 3H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842M / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711T / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266K / 267K / 437G / 486E / 522V / 5 69T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486F / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50M / 62L / 78E / 87E / 13 5Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 75 0P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 5 0V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486Y / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670R / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24M / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62F / 7 8E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711 H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 5 69Y / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711Q / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 13 5Q / 150S / 266N / 267K / 437G / 486I / 522V / 569T / 670T / 692G / 711H / 736M / 75 0P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 5 0V / 62L / 78E / 87E / 135H / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670H / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522N / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894E / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871Q / 883H / 894G / 913R / 932A、24W / 28E / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842N / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78H / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A 6E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62 L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 、 883H / 894G / 913I / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913H / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78 E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871Y / 883H / 894G / 913R / 932A, 24W / 28S / 29N / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 9 32A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 4 86E / 522V / 569T / 670K / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 88 3H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 2 66N / 267K / 437G / 486E / 522V / 569T / 670T / 692A / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50T / 62L / 78E / 8 7E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736 M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39 Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670 T / 692G / 711H / 736M / 750P / 812E / 830K / 842H / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692M / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29Q / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894D / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913F / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670D / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62M / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486C / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78T / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267H / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29I / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24H / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135I / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692H / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28H / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913N / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842D / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522K / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894H / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78K / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39F / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871W / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Y / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842Q / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29H / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78Q / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522D / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932Y、24W / 28S / 29V / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S、 / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842L / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692F / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29R / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 84 2S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 8 7E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894I / 913R / 932A, 24W / 28S / 29T / 39N / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150 S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62 L / 78E / 87E / 135Q / 150S / 266E / 267K / 437G / 486E / 522V / 569T / 670T / 69 2G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78C / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932P、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932D、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830Y / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135K / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692D / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28G / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711N / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486W / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50D / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486A / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894N / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932G、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711D / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871H / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486H / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913K / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871M / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39E / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932N、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932M、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913M / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569W / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486S / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711I / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711C / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50W / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522C / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 8、 71E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R、24W / 28S / 29W / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812G / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78D / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871D / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692I / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29F / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522M / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670N / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29M / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486G / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932K、24W / 28S / 29T / 39Q / 50V / 62Q / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830S / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812S / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830G / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486M / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711K / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932H、24W / 28S / 29P / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692W / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29Y / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692C / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29G / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812D / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932C、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871I / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692Y / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750Q / 812E / 830K / 842 S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 71 1H / 736M / 750P / 812E / 830H / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 4 86E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842C / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871S / 883H / 894G / 913R / 932A, 24W / 28 S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871C / The amino acid positions include 883H / 894G / 913R / 932A, or 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913W / 932A, and the amino acid positions correspond to the reference sequence corresponding to residues 20-944 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2.

[0154] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase includes a sequence containing residues 20-944 of even-numbered sequence numbers 14-754, or a sequence containing even-numbered sequence numbers 14-754.

[0155] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase is as follows: SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136 ,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 2 62, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 3 24, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 38 6, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448 ,450,452,454,456,458,460,462,464,466,468,470,472,474,476,478,480,482,484,486,488,490,492,494,496,498,500,502,504,506,508,510,512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 5 74, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636 ,638,640,642,644,646,648,650,652,654,656,658,660,662,664,666,668,670,672,674,676,678,680,682,684,686,688,690,692,694,696,698,7 The amino acid sequence includes residues 20-944 of 00, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, or 754. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid insertions, deletions, or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions. In some embodiments, the amino acid sequence optionally has one, two, three, four, or up to five amino acid insertions, deletions, or substitutions. In some embodiments, the amino acid sequence optionally has one, two, three, four, or up to five amino acid substitutions. In some embodiments, the amino acid substitutions include non-conservative or conservative substitutions. In some embodiments, the amino acid substitutions include conservative substitutions. In some embodiments, guidance regarding non-conservative and conservative substitutions is provided by the modifications and examples disclosed herein.

[0156] In some embodiments, the amino acid sequence of the manipulated acid alpha-glucosidase is as follows: SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136 ,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 2 62, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 3 24, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 38 6, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448 ,450,452,454,456,458,460,462,464,466,468,470,472,474,476,478,480,482,484,486,488,490,492,494,496,498,500,502,504,506,508,510,512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572 , 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634 , including 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, or 754. In some embodiments, the amino acid sequence optionally has one, two, three, four, five, six, seven, eight, nine, or up to ten amino acid insertions, deletions, or substitutions. In some embodiments, the amino acid sequence optionally has one, two, three, four, five, six, seven, eight, nine, or up to ten amino acid substitutions. In some embodiments, the amino acid sequence optionally has one, two, three, four, or up to five amino acid insertions, deletions, or substitutions. In some embodiments, the amino acid sequence optionally has one, two, three, four, or up to five amino acid substitutions. In some embodiments, the amino acid substitutions include non-conservative or conservative substitutions. In some embodiments, the amino acid substitutions include conservative substitutions. In some embodiments, guidance regarding non-conservative and conservative substitutions is provided by the variations and examples disclosed herein.

[0157] In some embodiments, the manipulated acid alpha-glucosidases of the present disclosure have acid alpha-glucosidase activity. In some embodiments, the manipulated acid alpha-glucosidases have acid alpha-glucosidase activity and at least one improved or enhanced property compared to a reference acid alpha-glucosidase.

[0158] In some embodiments, the manipulated acid alpha-glucosidase is more thermally stable than the reference acid alpha-glucosidase.

[0159] In some embodiments, the manipulated acid alpha-glucosidase is more stable at pH 7 (e.g., at neutral pH) than the reference acid alpha-glucosidase.

[0160] In some embodiments, the manipulated acid alpha-glucosidase is more stable than the reference acid alpha-glucosidase at acidic pH, particularly around pH 4.4.

[0161] In some embodiments, the manipulated acid alpha-glucosidase shows increased expression compared to the reference acid alpha-glucosidase.

[0162] In some embodiments, the manipulated acid alpha-glucosidase is more stable within liposomes than the reference acid alpha-glucosidase.

[0163] In some embodiments, the manipulated acid alpha-glucosidase is more stable in plasma, particularly in human plasma, than the reference acid alpha-glucosidase.

[0164] In some embodiments, the manipulated acid alpha-glucosidase is more readily taken up by human cells than the reference acid alpha-glucosidase.

[0165] In some embodiments, the manipulated acid alpha-glucosidase exhibits greater enzymatic activity in the cell lysate than the reference acid alpha-glucosidase.

[0166] In some embodiments, the manipulated acid alpha-glucosidase exhibits reduced immunogenicity compared to the reference acid alpha-glucosidase.

[0167] In some embodiments, the reference acid alpha-glucosidase has a sequence corresponding to residues 20-944 of SEQ ID NO: 2 or 12, or a sequence corresponding to SEQ ID NO: 2 or 12. In some embodiments, the reference acid alpha-glucosidase has a sequence corresponding to residues 20-944 of SEQ ID NO: 2, or a sequence corresponding to SEQ ID NO: 2. In some embodiments, the reference acid alpha-glucosidase has a sequence corresponding to residues 20-944 of SEQ ID NO: 12, or a sequence corresponding to SEQ ID NO: 12. Exemplary improved properties are provided in the examples.

[0168] In some embodiments, the manipulated acid alpha-glucosidase exhibits, compared to the reference acid alpha-glucosidase, i) improved catalytic activity, ii) increased tolerance to pH 7, iii) increased tolerance to pH 4.4, iv) increased stability within lysosomes, v) increased expression, vi) increased uptake into cells, vii) increased enzyme activity in cell lysates, viiii) increased stability in plasma / serum, and ix) decreased immunogenicity, or at least one improved property selected from any combination of i), ii), iii), iv), v), vi), vii), viiii), and ix). In some embodiments, the reference acid alpha-glucosidase has a sequence corresponding to residues 20-944 of SEQ ID NO: 2 or 12, or a sequence corresponding to SEQ ID NO: 2 or 12. In some embodiments, the reference acid alpha-glucosidase has a sequence corresponding to residues 20-944 of SEQ ID NO: 12, or a sequence corresponding to SEQ ID NO: 12.

[0169] In some embodiments, the manipulated acid alpha-glucosidase exhibits reduced immunogenicity compared to a reference acid alpha-glucosidase having a sequence corresponding to residues 20-944 of SEQ ID NO: 2 or 12, or a sequence corresponding to SEQ ID NO: 2 or 12. In some embodiments, the manipulated acid alpha-glucosidase exhibits a decrease in total immunogenicity score (TIS) of more than 10, more than 100, or more than 200 compared to the reference acid alpha-glucosidase of SEQ ID NO: 2. In some embodiments, the manipulated acid alpha-glucosidase exhibits a decrease in immunogenicity hit count (IHC) of more than 2, more than 5, or more than 20 compared to the reference acid alpha-glucosidase of SEQ ID NO: 2. In some embodiments, the manipulated acid alpha-glucosidase exhibits a decrease in total immunogenicity score (TIS) of more than 10, more than 100, or more than 200 compared to the reference acid alpha-glucosidase of SEQ ID NO: 12. In some embodiments, the manipulated acid alpha-glucosidases exhibit a reduction in immunogenicity hit count (IHC) of more than 2, more than 5, or more than 20 compared to the reference acid alpha-glucosidase of SEQ ID NO: 12. Exemplary manipulated acid alpha-glucosidase polypeptides exhibiting reduced immunogenicity based on TIS and / or IHC can be selected from the manipulated acid alpha-glucosidases presented in Table 4-1 of the Examples.

[0170] In some embodiments, the manipulated acid alpha-glucosidase polypeptides, as provided in the Examples, exhibit a smaller number of sequence regions presented by antigen-presenting cells and / or a reduced frequency of peptide presentation in MHC-related peptide proteomics (MAPPs) assays compared with the reference acid alpha-glucosidase polypeptide, particularly the reference acid alpha-glucosidase polypeptide corresponding to SEQ ID NO: 2.

[0171] In some embodiments, the engineered acid alpha-glucosidase described herein comprises a prepropeptide of the engineered acid alpha-glucosidase. In some embodiments, the prepropeptide of the engineered acid alpha-glucosidase comprises a eukaryotic or synthetic signal peptide sequence. In some embodiments, the signal peptide of the prepropeptide of the engineered acid alpha-glucosidase comprises a mouse or human signal peptide sequence. In some embodiments, the signal peptide comprises a sequence containing residues 1-19 of SEQ ID NO: 2 or 12.

[0172] In some embodiments, the manipulated acid alpha-glucosidase comprises a propolypeptide of the manipulated acid alpha-glucosidase described herein. In some embodiments, the propolypeptide of the manipulated acid alpha-glucosidase lacks a signal sequence. In some embodiments, the propolypeptide of the manipulated acid alpha-glucosidase comprises residues 20-944 of the manipulated acid alpha-glucosidase described herein.

[0173] In some embodiments, the engineered acid alpha-glucosidase is the mature form of the engineered acid alpha-glucosidase polypeptide described herein. In some embodiments, the mature form of the engineered acid alpha-glucosidase is the secretory form of the engineered acid alpha-glucosidase.

[0174] In some embodiments, the engineered acid alpha-glucosidase further comprises a fusion polypeptide. In some embodiments, the engineered acid alpha-glucosidase polypeptide described herein can be fused to a variety of polypeptide sequences, such as polypeptide tags, which can be used for detection and / or purification, as an example, but are not limited. In some embodiments, the fusion protein of the engineered acid alpha-glucosidase polypeptide comprises a glycine-histidine or histidine tag (His tag). In some embodiments, the fusion protein of the engineered acid alpha-glucosidase polypeptide comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In some embodiments, the fusion protein of the engineered acid alpha-glucosidase polypeptide comprises a GST, SUMO, Strep, MBP, or GFP tag. In some embodiments, the fusion is to the amino (N) terminus of the engineered acid alpha-glucosidase polypeptide. In some embodiments, the fusion is to the carboxy (C) terminus of the engineered acid alpha-glucosidase polypeptide. In some embodiments, the fusion polypeptide is inserted after the signal sequence and before the manipulated acid alpha-glucosidase polypeptide, enabling the expression and secretion of the polypeptide containing the fusion polypeptide (e.g., polypeptide tag) and the polypeptide.

[0175] In some embodiments, the manipulated acid alpha-glucosidase is an isolated or purified preparation. In some embodiments, the manipulated acid alpha-glucosidase is purified from a mixture (e.g., from cells or cell cultures) using one or more of the known techniques for protein purification, including, in particular, lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography.

[0176] Chromatographic techniques for the isolation and purification of engineered acid alpha-glucosidase polypeptides include, in particular, reverse-phase chromatography, high-performance liquid chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, gel electrophoresis, and affinity chromatography. The conditions for purifying a particular protein depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, and molecular shape, and will be apparent to those skilled in the art. In some embodiments, affinity techniques may be used to isolate the polypeptide. For affinity chromatography purification, any antibody that specifically binds to the acid alpha-glucosidase polypeptide of interest may be found to be useful. For antibody production, various host animals, including but not limited to rabbits, mice, rats, and camelids, are immunized by injecting the engineered acid alpha-glucosidase polypeptide or a fragment thereof.

[0177] In some embodiments, the Disclosure further provides functional or biologically active fragments of the engineered acid alpha-glucosidase polypeptide described herein. Thus, for each and all embodiments of the engineered acid alpha-glucosidase described herein, a functional or biologically active fragment of the engineered acid alpha-glucosidase is provided herein together with the engineered acid alpha-glucosidase. In some embodiments, the functional or biologically active fragment of the engineered acid alpha-glucosidase contains at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the activity of the acid alpha-glucosidase polypeptide from which it is derived (i.e., the acid alpha-glucosidase).

[0178] In some embodiments, the functional or biologically active fragment of the manipulated acid alpha-glucosidase described herein comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the manipulated acid alpha-glucosidase. In some embodiments, the functional fragment may be cleaved by fewer than 5, 10, 15, 10, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 amino acids.

[0179] In some embodiments, the functional or biologically active fragments of the manipulated acid alpha-glucosidase polypeptide described herein contain at least one substitution or set of substitutions in the amino acid sequence of the manipulated acid alpha-glucosidase described herein. Thus, in some embodiments, the functional or biologically active fragment(s) of the manipulated acid alpha-glucosidase exhibits an improvement or enhancement of the properties related to the substitution or set of substitutions in the acid alpha-glucosidase.

[0180] Polynucleotides encoding recombinant polypeptides, expression vectors, and host cells In another embodiment, the disclosure provides recombinant polynucleotides encoding the engineered acid alpha-glucosidase polypeptide described herein. In some embodiments, the polynucleotides are operably linked to one or more heterologous or homologous regulatory sequences that control gene expression to create recombinant polynucleotides capable of expressing the polypeptide. An expression construct containing the heterologous polynucleotide encoding the engineered acid alpha-glucosidase polypeptide can be introduced into a suitable host cell to express the corresponding engineered acid alpha-glucosidase polypeptide.

[0181] As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of codons corresponding to various amino acids provides a description of all polynucleotides that can encode the polypeptide of the subject. The degeneracy of the genetic code, in which the same amino acids are encoded by alternative or synonymous codons, makes it possible to create a very large number of nucleic acids, all of which encode the engineered acid alpha-glucosidase polypeptide. Thus, with knowledge of a particular amino acid sequence, those skilled in the art can create any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not alter the amino acid sequence of the protein. In this regard, this disclosure specifically considers each and all possible variations of polynucleotides that can encode the engineered acid alpha-glucosidase polypeptide described herein by selecting combinations based on the selection of possible codons, and all such variations should be considered specifically disclosed for any polypeptide described herein, including the engineered acid alpha-glucosidase variants provided in the Examples.

[0182] In some embodiments, recombinant polynucleotides are codon-optimized. In other words, codons are preferably selected to fit the host cell in which the protein is produced. For example, preferred codons used in bacteria are used for expression in bacteria, preferred codons used in fungi are typically used for expression in fungi, and preferred codons used in mammals are used for expression in mammals and mammalian cells. In some embodiments, codon-optimized polynucleotides encoding an engineered acid alpha-glucosidase polypeptide contain preferred codons at about 40%, 50%, 60%, 70%, 80%, or more than 90% of the codon positions in the full-length coding region. In some embodiments, the present disclosure provides recombinant polynucleotide sequences in which codons are optimized for expression in human cells or tissues.

[0183] As stated above, it should be understood that this disclosure provides recombinant polynucleotides encoding each and all of the manipulated acid alpha-glucosidase polypeptides described herein. Accordingly, as an example, but not limited to, in some embodiments, the recombinant polynucleotides of the present disclosure include a polynucleotide sequence encoding an engineered acid alpha-glucosidase polypeptide or a biologically active fragment thereof, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity with respect to a reference sequence corresponding to residues 20-944 of even-numbered sequence codes 2, 12, and 14-754, or with respect to an even-numbered sequence code 2, 12, and 14-754, wherein the amino acid sequence comprises one or more substitutions with respect to a reference sequence corresponding to residues 20-944 of sequence code 12 or 2, or with respect to a reference sequence corresponding to sequence code 12 or 2.

[0184] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acid alpha-glucosidase, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity with respect to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12 or 2, or to a reference sequence corresponding to SEQ ID NO: 12 or 2, wherein the amino acid sequence comprises one or more substitutions with respect to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12 or 2, or to a reference sequence corresponding to SEQ ID NO: 12 or 2.

[0185] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acid alpha-glucosidase, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity with respect to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12, or to a reference sequence corresponding to SEQ ID NO: 12, wherein the amino acid sequence comprises one or more substitutions with respect to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12, or to a reference sequence corresponding to SEQ ID NO: 12.

[0186] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acid alpha-glucosidase, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity with respect to a reference sequence corresponding to even-numbered sequence numbers 14-754, wherein the amino acid sequence comprises one or more substitutions with respect to a reference sequence corresponding to residues 20-944 of sequence number 12 or 2, or to a reference sequence corresponding to sequence number 12 or 2.

[0187] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acid alpha-glucosidase, comprising an amino acid sequence having at least one substitution at amino acid positions 24, 28, 29, 39, 50, 62, 78, 87, 135, 150, 266, 267, 305, 437, 486, 522, 569, 670, 692, 711, 736, 750, 812, 830, 842, 871, 883, 894, 913, or 932, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12, or to a reference sequence corresponding to SEQ ID NO: 12.

[0188] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence having at least one substitution at amino acid position 305.

[0189] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence having at least one substitution at amino acid position 24.

[0190] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence having at least one substitution at amino acid position 28.

[0191] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence having at least one substitution at amino acid position 29.

[0192] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence having at least one substitution at amino acid position 39.

[0193] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence having at least one substitution at amino acid position 50.

[0194] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence that includes at least one substitution at amino acid position 62.

[0195] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence that includes at least one substitution at amino acid position 78.

[0196] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence that includes at least one substitution at amino acid position 87.

[0197] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence having at least one substitution at amino acid position 135.

[0198] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence having at least one substitution at amino acid position 150.

[0199] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence having at least one substitution at amino acid position 266.

[0200] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence that includes at least one substitution at amino acid position 267.

[0201] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence that includes at least one substitution at amino acid position 437.

[0202] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence that includes at least one substitution at amino acid position 486.

[0203] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence that includes at least one substitution at amino acid position 522.

[0204] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence having at least one substitution at amino acid position 569.

[0205] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence having at least one substitution at amino acid position 670.

[0206] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence that includes at least one substitution at amino acid position 692.

[0207] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence having at least one substitution at amino acid position 711.

[0208] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence that includes at least one substitution at amino acid position 736.

[0209] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence having at least one substitution at amino acid position 750.

[0210] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence having at least one substitution at amino acid position 812.

[0211] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence having at least one substitution at amino acid position 830.

[0212] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence that includes at least one substitution at amino acid position 842.

[0213] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence having at least one substitution at amino acid position 305.

[0214] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence that includes at least one substitution at amino acid position 883.

[0215] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence that includes at least one substitution at amino acid position 894.

[0216] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence that includes at least one substitution at amino acid position 913.

[0217] In some embodiments, the recombinant polynucleotide encodes an engineered acid alpha-glucosidase comprising an amino acid sequence that includes at least one substitution at amino acid position 932.

[0218] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acid alpha-glucosidase having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity with respect to an engineered acid alpha-glucosidase having substitutions or sets of substitutions as shown in Table 3-1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12 or 2, or to a reference sequence corresponding to SEQ ID NO: 12 or 2.

[0219] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acid alpha-glucosidase, comprising an amino acid sequence having at least one substitution as shown in Table 3-1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12 or 2, or to a reference sequence corresponding to SEQ ID NO: 12 or 2.

[0220] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acid alpha-glucosidase, comprising an amino acid sequence containing at least the substitution set shown in Table 3-1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12 or 2, or to a reference sequence corresponding to SEQ ID NO: 12 or 2.

[0221] In some embodiments, recombinant polynucleotides have amino acid positions 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 305 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 871 / 883 / 894 / 913 / 932, 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 670 / 692 / 711 / 73 6 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 8 71 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932,24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842S / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、The polynucleotide sequence encoding an engineered acid alpha-glucosidase comprises an amino acid sequence containing at least a substitution set in 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913, or 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812S / 830 / 842 / 871 / 883 / 894 / 913 / 932, with the position relative to SEQ ID NO: 2. ,

[0222] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acid alpha-glucosidase, comprising residues 20-944 of even-numbered sequence numbers 14-754, or an amino acid sequence comprising a sequence comprising even-numbered sequence numbers 14-754.

[0223] In some embodiments, recombinant polynucleotides are sequence numbers 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 14 4, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206 ,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 3 32, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 39 4, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456 ,458,460,462,464,466,468,470,472,474,476,478,480,482,484,486,488,490,492,494,496,498,500,502,504,506,508,510,512,514,516,518,520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 5 86, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652 It contains a polynucleotide sequence encoding an engineered acid alpha-glucosidase, comprising an amino acid sequence containing residues 20-944 of 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, or 754.

[0224] In some embodiments, recombinant polynucleotides are sequence numbers 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 14 4, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206 ,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 3 32, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 39 4, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456 ,458,460,462,464,466,468,470,472,474,476,478,480,482,484,486,488,490,492,494,496,498,500,502,504,506,508,510,512,514,516,518,520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584 , 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 65 The polynucleotide sequence encoding an engineered acid alpha-glucosidase includes an amino acid sequence containing 0, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, or 754.

[0225] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity with respect to a reference polynucleotide sequence corresponding to nucleotide residues 58–2832 of odd-numbered sequence numbers 13–753, or with respect to a reference polynucleotide sequence corresponding to odd-numbered sequence numbers 13–753, wherein the recombinant polynucleotide encodes an acid alpha-glucosidase.

[0226] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence containing nucleotide residues 58 to 2832 of odd-numbered sequence numbers 13 to 753, or a polynucleotide sequence containing odd-numbered sequence numbers 13 to 753.

[0227] In some embodiments, recombinant polynucleotides are SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 14 3, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205 ,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 3 31, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 39 3, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455 ,457,459,461,463,465,467,469,471,473,475,477,479,481,483,485,487,489,491,493,495,497,499,501,503,505,507,509,511,513,515,517,519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 58 1, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 6 Contains a polynucleotide sequence including nucleotide extensions 58-2832 of 45, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, or 753.

[0228] In some embodiments, recombinant polynucleotides are SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 14 3, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205 ,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 3 31, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 39 3, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455 ,457,459,461,463,465,467,469,471,473,475,477,479,481,483,485,487,489,491,493,495,497,499,501,503,505,507,509,511,513,515,517,519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 57 9, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 6 Contains polynucleotide sequences including 41, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, or 753.

[0229] In some embodiments, recombinant polynucleotides encoding any of the manipulated acid alpha-glucosidase polypeptides provided herein are manipulated in various ways to provide expression of the encoded polypeptide. In some embodiments, the polypeptide-encoding polynucleotide is provided as an expression vector having one or more regulatory sequences to modulate the expression of the polynucleotide and / or polypeptide. In some embodiments, the regulatory sequences include, among other sequences, promoters, Kozak sequences, reader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and DNA-based regulatory elements for gene therapy retention and transcription termination factors. Manipulating isolated polynucleotides before insertion into a vector may be desirable or required, depending on the expression vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art.

[0230] In some embodiments, a suitable promoter can be selected based on the host cell used for expression. For bacterial host cells, suitable promoters for directing the transcription of the nucleic acid constructs of this disclosure include, but are not limited to, the E. coli lac operon, the agarase gene of Streptomyces coelicolor (dagA), the levans sucrase gene of Bacillus subtilis (sacB), the alpha-amylase gene of Bacillus licheniformis (amyL), the maltogenic amylase gene of Bacillus stearothermophilus (amyM), the alpha-amylase gene of Bacillus amyloliquefaciens (amyQ), the penicillinase gene of Bacillus licheniformis (penP), the xylA and xylB genes of Bacillus subtilis, and the beta-lactamase genes of prokaryotes (e.g., Villa-Kamaroff et al., Proc. Natl). Examples include promoters derived from tac promoters (see Acad.Sci.USA,1978,75:3727-3731) and tac promoters (see, for example, DeBoer et al., Proc.Natl Acad.Sci.USA,1983,80:21-25).Exemplary promoters of filamentous fungal host cells include, but are not limited to, TAKA amylase of Aspergillus oryzae, aspartate proteinase of Rhizomucor miehei, neutral alpha-amylase of Aspergillus niger, acid-stable alpha-amylase of Aspergillus niger, glucoamylase (glaA) of Aspergillus niger or Aspergillus awamori, lipase of Rhizomucor miehei, alkaline protease of Aspergillus oryzae, triose phosphate isomerase of Aspergillus oryzae, acetamidase of Aspergillus nidulans, and trypsin-like protease of Fusarium oxysporum (see, e.g., WO96 / 00787), as well as the NA2-tpi promoter (neutral alpha-amylase of Aspergillus niger and Aspergillus Examples include promoters derived from the gene for the triose phosphate isomerase of Saccharomyces cerevisiae (hybrid promoters), as well as their variants, cleaved forms, and hybrid promoters. Exemplary yeast cell promoters may be derived from the genes for enolase (ENO-1), galactokinase (GAL1), alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and 3-phosphoglycerate kinase of Saccharomyces cerevisiae. Other useful promoters for yeast host cells are known in the art (see, for example, Romanos et al., Yeast, 1992, 8:423-488).Examples of promoters for use in mammalian cells include, but are not limited to, those derived from cytomegalovirus (CMV), chicken β-actin promoter fused with CMV enhancer, monkey virus 40 (SV40), homosapiens phosphoglycerate kinase, beta-actin, elongation factor-1a or glyceraldehyde-3-phosphate dehydrogenase, or gallus β-actin.

[0231] In some embodiments, the regulatory sequence is a preferred transcription termination factor sequence, which is recognized by the host cell to terminate transcription. The termination factor sequence is operably ligated to the 3' end of the nucleic acid sequence encoding the polypeptide. Any termination factor that is functional in a selected host cell has been found to be of use in the present invention. In bacterial expression, the transcription termination factor can be a Rho-dependent termination factor that does not require a transcription factor, or a Rho-independent or endogenous termination factor. Exemplary bacterial transcription termination factors are described in Peters et al., J Mol Biol., 2011, 412(5):793-813. Exemplary transcription termination factors for filamentous fungal host cells can be obtained from genes for TAKA amylase of Aspergillus oryzae, glucoamylase of Aspergillus niger, anthranilate synthase of Aspergillus nidulans, alpha-glucosidase of Aspergillus niger, and trypsin-like protease of Fusarium oxysporum. Exemplary terminators for yeast host cells can be obtained from genes for enolase of Saccharomyces cerevisiae, cytochrome C (CYC1) of Saccharomyces cerevisiae, and glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Other useful termination factors for yeast host cells are known in the art (e.g., Romanos et al., see above). Examples of termination factors in mammalian cells include, but are not limited to, cytomegalovirus (CMV), monkey vacuolated virus 40 (SV40), Homo sapiens growth hormone (hGH), bovine growth hormone (BGH), and those derived from human or rabbit betaglobulin.

[0232] In some embodiments, the regulatory sequence is a preferred leader sequence, a 5'-cap modification, or a 5'UTR. In some embodiments, these regulatory sequence elements mediate binding to molecules involved in mRNA transport and translation, inhibit 5'-exonucleotide degradation, and confer resistance to cap removal. The leader sequence is operably ligated to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that functions in a selected host cell may be used. Exemplary leaders in filamentous fungal host cells are obtained from the TAKA amylase gene of Aspergillus oryzae and the triose phosphate isomerase gene of Aspergillus nidulans. Suitable leaders for yeast host cells include, but are not limited to, those derived from the genes of Saccharomyces cerevisiae: enolase (ENO-1), 3-phosphoglycerate kinase, alpha-factor, and alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP). Suitable leaders for mammalian host cells include, but are not limited to, the 5'-UTR element present in orthopoxvirus mRNA.

[0233] In some embodiments, the control sequence includes a 3' untranslated nucleic acid region and a polyadenylated tail nucleic acid sequence, a sequence operably ligated to the 3' end of a protein-coding nucleic acid sequence that mediates mRNA transport and translation, as well as binding to proteins involved in mRNA half-life. Any polyadenylated sequence and 3'UTR that are functional in selected host cells may be used in the present invention. Exemplary polyadenylated sequences in filamentous fungal host cells include, but are not limited to, TAKA amylase from Aspergillus oryzae, glucoamylase from Aspergillus niger, anthranilate synthase from Aspergillus nidulans, trypsin-like protease from Fusarium oxysporum, and alpha-glucosidase from Aspergillus niger. Useful polyadenylation sequences for yeast host cells are known in the art (see, for example, Guo and Sherman, Mol. Cell. Biol., 1995, 15:5983-5990). Useful polyadenylation and 3'UTR sequences for mammalian host cells include, but are not limited to, 3'-UTRs of α- and β-globin mRNA that have several sequence elements that increase mRNA stability and translation.

[0234] In some embodiments, the control sequence is a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of the polypeptide, thereby directing the coded polypeptide into the cell's secretory pathway. The 5' end of the coding sequence of the nucleic acid sequence may essentially contain a segment of the coding region encoding the secreted polypeptide and a signal peptide coding region natively linked within the translational reading frame. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. Any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a selected host cell has been found to be of use for the expression of the engineered acid alpha-glucosidase polypeptides provided herein. Effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, signal peptide coding regions derived from the genes for TAKA amylase of Aspergillus oryzae, neutral amylase of Aspergillus niger, glucoamylase of Aspergillus niger, aspartate proteinase of Rhizomucor miehei, cellulase of Humicola insolens, and lipase of Humicola lanuginosa. Useful signal peptides for yeast host cells include, but are not limited to, Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Useful signal peptides for mammalian host cells include, but are not limited to, those derived from the immunoglobulin gamma (IgG) gene. In some embodiments, the signal peptide contains a signal sequence naturally occurring within human acid alpha-glucosidase. In some embodiments, the encoded signal peptide contains a sequence including residues 1-19 of SEQ ID NO: 2 or 12.

[0235] In some embodiments, the regulatory sequence comprises one or more regulatory sequences that facilitate the regulation of the expression of polynucleotides and / or corresponding encoded polypeptides for host cell growth. Examples of regulatory systems include those that turn gene expression on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. In prokaryotic host cells, preferred regulatory sequences include, but are not limited to, the lac, tac, and trp operator systems. In yeast host cells, preferred regulatory systems include, but are not limited to, the ADH2 system or the GAL1 system. In filamentous fungi, preferred regulatory sequences include, but are not limited to, the TAKA alpha-amylase promoter, the glucoamylase promoter of Aspergillus niger, and the glucoamylase promoter of Aspergillus oryzae. Exemplary inducible promoters modulated by exogenous agents include the zinc-inducible sheep metallotionine (MT) promoter, the dexamethasone (Dex)-inducible promoter, the mouse mammary tumor virus (MMTV) promoter, the ecdysone insect promoter, the tetracycline-inducible promoter system, the RU486-inducible promoter system, and the rapamycin-inducible promoter system.

[0236] In some embodiments, the recombinant expression vector may be any suitable vector (including, but not limited to, adenoviruses (AV), adeno-associated viruses (AAV), lentiviruses (LV), and non-viral vectors such as liposomes and exosomes, plasmids, or viruses) that can conveniently carry out recombinant DNA procedures and result in the expression of the manipulated acid alpha-glucosidase polynucleotide sequence. The choice of vector typically depends on the compatibility of the vector with the host cell into which it is introduced. The vector may be a linear or closed circular plasmid.

[0237] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector existing as an extrachromosomal entity that replicates independently of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome). The vector may contain any means to ensure self-replication. In some alternative embodiments, the vector, upon introduction into a host cell, may integrate into the genome and replicate along with the chromosome(s) in which it is integrated. In some embodiments, the vector may be an episomal, non-replicating, and non-integrating vector. Furthermore, a single vector or plasmid, or two or more vectors or plasmids containing the total DNA to be introduced into the host cell's genome, or a transposon may be used.

[0238] In some embodiments, recombinant polynucleotides may be provided on a non-replicating expression vector or plasmid. In some embodiments, the non-replicating expression vector or plasmid may be based on a viral vector with replication defects (see, for example, Travieso et al., npj Vaccines, 2022, Vol. 7, Article 75).

[0239] In some embodiments, the expression vector contains one or more selectable markers that allow for easy selection of transformed cells. A “selectable marker” is a gene whose product confers resistance to biocides or viruses, heavy metal resistance, prototrophicity to trophic requirement strains, etc. Examples of bacterial selectable markers, but not limited to, include the dal gene from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. Suitable markers for yeast host cells, but not limited to, include ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase, e.g., from A. nidulans or A. orzyae), argB (ornithine carbamoyltransferase), bar (phosphinotrysin acetyltransferase, e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase, e.g., from A. nidulans or A. orzyae), sC (adenylate transferase), and trpC (anthranilate synthase), as well as their equivalents. Selectable markers for mammalian cells include, but are not limited to, chloramphenicol acetyltransferase (CAT), nurceotricin N-acetyltransferase, blasticidin-S deaminase, blasticidin S-acetyltransferase, Sh ble (Zeocin® resistance), aminoglycoside 3'-phosphotransferase (neomycin resistance), hph (hygromycin resistance), thymidine kinase, and puromycin N-acetyltransferase.

[0240] In another embodiment, the Disclosure provides a host cell comprising a recombinant polynucleotide encoding an engineered acid alpha-glucosidase polypeptide as described herein, wherein the polynucleotide is operably linked to one or more regulatory sequences for the expression of the engineered acid alpha-glucosidase polypeptide in the host cell. In some embodiments, the host cell is a prokaryotic or eukaryotic cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human cell. In some embodiments, the host cell is a cell lacking acid alpha-glucosidase activity or a cell obtained from a subject having Pompe disease.

[0241] Host cells for use in the expression of polypeptides encoded by the expression vectors of this disclosure include, but are not limited to, fungal cells, e.g., yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris (e.g., ATCC accession number 201178)); insect cells (e.g., Drosophila S2 and Spodoptera Sf9 cells); plant cells; animal cells (e.g., CHO, CHO-K1, COS, and BHK); and human cells (e.g., HEK293T, human fibroblasts, THP-1, Jurkat, and Bowes melanoma cell lines). In some embodiments, the host cells are cells obtained from mammals, including model animals or human patients lacking acid alpha-glucosidase activity. In some embodiments, the host cells for expression are cells obtained from human patients diagnosed with Pompe disease.

[0242] In another embodiment, the Disclosure provides a method for preparing an engineered acid alpha-glucosidase polypeptide, the method comprising culturing a host cell capable of expressing a polynucleotide encoding the engineered acid alpha-glucosidase polypeptide under conditions suitable for polypeptide expression such that the engineered acid alpha-glucosidase polypeptide is produced. In some embodiments, the method further comprises the step of isolating the engineered acid alpha-glucosidase polypeptide from, for example, a culture medium and / or cells. In some embodiments, the method further comprises purifying the expressed engineered acid alpha-glucosidase as described herein.

[0243] Appropriate culture media and growth conditions for the host cells described above are known in the art. Polynucleotides for the expression of the manipulated acid alpha-glucosidase polypeptide can be introduced into cells by various methods known in the art. These techniques include, in particular, electroporation, biolithographic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0244] In some embodiments, engineered acid alpha-glucosidase polypeptides having the properties disclosed herein can be obtained by performing mutagenesis and / or directed evolution techniques known in the art and described herein on polynucleotides encoding naturally occurring or engineered acid alpha-glucosidase polypeptides. An exemplary directed evolution technique is mutagenesis and / or DNA shuffling (see, for example, Stemmer, Proc. Natl. Acad. Sci. USA, 1994, 91:10747-10751; WO95 / 22625; WO97 / 0078; WO97 / 35966; WO98 / 27230; WO00 / 42651; WO01 / 75767 and U.S. Patent No. 6,537,746). Other directed evolutionary procedures that can be used include, in particular, the staggered extension process (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 1998, 16:258-261), mutagenic PCR (see, e.g., Caldwell et al., PCR Methods Appl., 1994, 3:S136-S140), and cassette mutagenesis (see, e.g., Black et al., Proc. Natl. Acad. Sci. USA, 1996, 93:3525-3529).

[0245] Mutagenic and directed evolution methods can be applied to polynucleotides to generate variant libraries that can be expressed, screened, and assayed. Any suitable mutagenic and directed evolution method found to be used in this disclosure (e.g., U.S. Patents No. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,277,638, 6,287,861, 6, 287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883, 6,319,713, 6,319,714, 6,323,030, 6,326,204, No. 6,335,160, No. 6,335,198, No. 6,344,356, No. 6,352,859, No. 6,355, 484, 6,358,740, 6,358,742, 6,365,377, 6,365,408, No. 6,368,861, No. 6,372,497, No. 6,376,246, No. 6,379,964, No. 6,387,702, No. 6,391,552, No. 6,391,640, No. 6,395,547, No. 6,406,855 , No. 6,406,910, No. 6,413,745, No. 6,413,774, No. 6,420,175, No. 6,4 No. 23,542, No. 6,426,224, No. 6,436,675, No. 6,444,468, No. 6,455,253 No. 6,479,652, No. 6,482,647, No. 6,489,146, No. 6,506,602, No. 6,506,603, No. 6,519,065, No. 6,521,453, No. 6,528,311, No. 6,537,7 No. 46, No. 6,573,098, No. 6,576,467, No. 6,579,678, No. 6,586,182, No. No. 6,602,986, No. 6,613,514, No. 6,653,072, No. 6,716,631, No. 6,946,No. 296, No. 6,961,664, No. 6,995,017, No. 7,024,312, No. 7,058,515, No. 7,105,297, No. 7,148,054, No. 7,288,375, No. 7,421,347, No. 7,430,477, No. 7,534,564, No. 7,620,500, No. 7,620,502, No. 7,629,170, No. 7,702,464, No. 7,747,391, No. 7,747,393, No. 7,751,986, No. 7,776,598, No. 7,783,428, No. 7,795,030, No. 7,853,410, No. 7,868,138, No. 7,873,499, No. 7,904,249, No. 7,957,912, No. 8,383,346, No. 8,504,498, No. 8,849,575, No. 8,876,066, No. 8,768,871, No. 9,593,326, and all related non-US counterpart documents; Ling et al., Anal. Biochem., 1997, 254(2):157-78; Dale et al., Meth. Mol. Biol., 1996, 57:369-74; Smith, Ann. Rev. Genet., 1985, 19:423-462; Botstein et al., Science, 1985, 229:1193-1201; Carter, Biochem. J., 1986, 237:1-7; Kramer et al., Cell, 1984, 38:879-887; Wells et al., Gene, 1985, 34:315-323; Minshull et al., Curr. Op. Chem. Biol., 1999, 3:284-290; Christians et al., Nat. Biotechnol., 1999, 17:259-264; Crameri et al., Nature, 1998, 391:288-291; Crameri, et al., Nat. Biotechnol., 1997, 15:436-438; Zhang et al., Proc. Nat. Acad. Sci. U.S.A., 1997, 94:4504-4509; Crameri et al., Nat. Biotechnol., 1996, 14:315-319; Stemmer, Nature,1994, 370:389-391; Stemmer, Proc. Nat. Acad. Sci. USA, 1994, 91:10747-10751; U.S. Patent Application Publications 2008 / 0220990, 2009 / 0312196, 2014 / 0005057, 2014 / 0214391, 2014 / 0221216; 2015 / 00506 See issues 58, 2015 / 0133307, 2015 / 0134315, and all related non-U.S. corresponding documents; see WO95 / 22625, WO97 / 0078, WO97 / 35966, WO98 / 27230, WO00 / 42651, WO01 / 75767, WO2009 / 152336 (all of which are incorporated herein by reference).

[0246] In some embodiments, polypeptide variants obtained after mutagenesis treatment are screened by subjecting the polypeptide variants to defined assay conditions such as temperature, acidic or basic pH, and serum / plasma exposure, and measuring the amount of polypeptide activity remaining after the treatment or other assay conditions. The DNA containing the polynucleotide encoding the manipulated acid alpha-glucosidase polypeptide is then isolated from host cells, sequenced to identify (if present) changes in the nucleotide sequence, and used to express the protein in different host cells or the same host cells. Measurement of activity from the expression library can be performed using any preferred method known in the art, and also as provided in the examples.

[0247] For manipulated polypeptides of known sequences, the polynucleotides encoding the polypeptides can be prepared by standard solid-phase methods according to known synthetic methods. In some embodiments, polynucleotide fragments can be synthesized individually and then joined (e.g., by enzymatic or chemical litigation, or polymerase-mediated methods) to form any desired continuous sequence. For example, the polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classical phosphoramidite method (see, e.g., Beaucage et al., Tetra. Lett., 1981, 22:1859-69, and Matthes et al., EMBO J., 1984, 3:801-05), as typically carried out by automated synthesis methods. According to the phosphoramidite method, oligonucleotides are synthesized (e.g., in an automated DNA synthesizer), purified, annealed, ligated, and cloned in a suitable vector.

[0248] Accordingly, in some embodiments, a method for preparing an engineered acid alpha-glucosidase polypeptide may comprise (a) synthesizing an engineered acid alpha-glucosidase, e.g., any variant provided in Table 3-1, and a polynucleotide encoding an amino acid sequence of even-numbered sequence numbers 14-754, and (b) expressing the engineered acid alpha-glucosidase polypeptide encoded by the polynucleotide. In some embodiments, the amino acid sequence optionally has deletions, insertions, and / or substitutions of 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residues. In some embodiments, the amino acid sequence optionally has deletions, insertions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residues. In some embodiments, the amino acid sequence optionally has deletions, insertions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residues. In some embodiments, the substitutions are either conserved or non-conservative substitutions.

[0249] The expressed and manipulated acid alpha-glucosidase polypeptides can be evaluated for any desired improved properties (e.g., activity / potency, stability, serum / plasma stability, basic pH tolerance, acid pH tolerance, cell uptake, etc.) using any suitable assay known in the art, including but not limited to the assays and conditions described herein.

[0250] composition In further embodiments, the Disclosure provides compositions comprising an engineered acid alpha-glucosidase or a recombinant polynucleotide encoding an acid alpha-glucosidase, the compositions including, but not limited to, those described below. In some embodiments, the compositions comprise at least an engineered acid alpha-glucosidase or recombinant polynucleotide as illustrated in Tables 3-1 and 4-1, and in the sequence listings.

[0251] Depending on the composition and mode of administration, the compositions comprising the manipulated acid alpha-glucosidase described herein may be in solid, semi-solid, or liquid form. In some embodiments, the compositions may include other pharmaceutically acceptable components such as diluents, buffers, excipients, salts, emulsifiers, preservatives, stabilizers, fillers, and other components. In some embodiments, the compositions comprising the manipulated acid alpha-glucosidase polypeptide of the Disclosure may include one or more commonly used carrier compounds, including but not limited to sugars (e.g., lactose, sucrose, mannitol, and / or sorbitol), starch, cellulose (e.g., methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose), gums (e.g., arabic, tragacanth, guar, etc.), and / or proteins (e.g., gelatin, collagen, etc.). Technical details for formulation and administration are available in the art and described in the literature.

[0252] In some embodiments, the manipulated acid alpha-glucosidase polypeptide is formulated for use in pharmaceutical compositions. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient. Any suitable form for use in the delivery of the manipulated acid alpha-glucosidase polypeptide, including but not limited to pills, tablets, gel tabs, capsules, lozenges, sugar-coated tablets, powders, soft gels, sol gels, gels, emulsions, implants, patches, sprays, ointments, topical medications, creams, pastes, jellies, paints, aerosols, chewing gums, analgesics, sticks, solutions, suspensions (including, but not limited to, oily suspensions, water-in-oil emulsions, etc.), slurries, syrups, controlled-release formulations, etc., is found to be used herein. In some embodiments, the manipulated acid alpha-glucosidase polypeptide is provided in a form suitable for injection or infusion (i.e., injectable formulation), particularly for parenteral administration or infusion to patients, particularly human patients.

[0253] In some embodiments, the engineered acid alpha-glucosidase polypeptide is provided in a biocompatible matrix such as a sol gel, including a silica-based (e.g., oxysilane) sol gel. In some embodiments, the engineered acid alpha-glucosidase polypeptide is encapsulated. In some alternative embodiments, the engineered acid alpha-glucosidase polypeptide is encapsulated in nanostructures (e.g., nanotubes, nanotubular structures, nanocapsules, or microcapsules, microspheres, liposomes, etc.). The engineered acid alpha-glucosidase polypeptide is intended to be administered by any preferred means known in the art, including but not limited to parenteral (e.g., intravenous, intramuscular, subcutaneous, etc.), oral, topical, transdermal, intranasal, intraocular, subarachnoid, via implants, etc.

[0254] In some embodiments, the manipulated acid alpha-glucosidase polypeptide is modified by glycosylation, chemical crosslinking, pegylation (i.e., by polyethylene glycol [PEG] or activated PEG, etc.) or other compounds (see, for example, Ikeda, Amino Acids, 2005, 29:283-287; U.S. Patents 7,531,341, 7,534,595, and 7,560,263; U.S. Patent Publications 2013 / 0039898 and 2012 / 0177722, etc.).

[0255] In some additional embodiments, the engineered acid alpha-glucosidase polypeptide is provided in a formulation comprising a matrix-stabilized enzyme crystal. In some embodiments, the formulation comprises a cross-linked crystal of the engineered acid alpha-glucosidase enzyme and a polymer having a reactive moiety that adheres to the enzyme crystal. The present invention also provides engineered acid alpha-glucosidase polypeptide in a polymer.

[0256] In some embodiments, compositions comprising an engineered acid alpha-glucosidase polypeptide include, but are not limited to, one or more commonly used carrier compounds, including sugars (e.g., lactose, sucrose, mannitol, and / or sorbitol), starches (e.g., corn, wheat, rice, potato, or other plant starches), cellulose (e.g., methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose), gums (e.g., arabic, tragacanth, guar, etc.), and / or proteins (e.g., gelatin, collagen, etc.). Additional components in the oral formulation may include colorants and / or sweeteners (e.g., glucose, sucrose, and mannitol) and lubricants (e.g., magnesium stearate), as well as enteric coatings (e.g., methacrylate polymers, hydroxypropyl methylcellulose phthalate, and / or other suitable enteric coatings known in the art). In some embodiments, disintegrants or solubilizers (e.g., cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, e.g., sodium alginate) are included. In some embodiments, the manipulated acid alpha-glucosidase polypeptide is combined with a variety of additional components, including but not limited to preservatives, suspending agents, thickeners, wetting agents, alcohols, fatty acids, and / or emulsifiers, particularly in liquid formulations. In some embodiments, the manipulated acid alpha-glucosidase polypeptide is administered to a subject in combination with pharmacological chaperones and any other suitable compounds, molecules, and / or materials used in the treatment of Pompe disease, including but not limited to these.

[0257] In some embodiments, the pharmaceutical composition comprises a recombinant polynucleotide encoding the engineered acid alpha-glucosidase described herein. In some embodiments, the recombinant polynucleotide is DNA or mRNA. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient suitable for administration of the recombinant polynucleotide. In some embodiments, the pharmaceutical composition comprising the recombinant polynucleotide encoding the engineered acid alpha-glucosidase is suitable for any preferred means of administration, including intravenous, intramuscular, subcutaneous, oral, intranasal, intraocular, intrathecal, and inhalation.

[0258] In some additional embodiments, recombinant polynucleotides encoding engineered acid alpha-glucosidase polypeptides are provided for delivery to cells or tissues via gene therapy, particularly including viral delivery vectors, including adenovirus (AV), adeno-associated virus (AAV), and lentivirus (LV). In some embodiments, recombinant polynucleotides encoding engineered acid alpha-glucosidase are provided for delivery in non-viral vectors or formulations, particularly including liposomes, nanotubes, nanotubular structures, nanocapsules, or microcapsules, and microspheres. In some embodiments, recombinant polynucleotides encoding engineered acid alpha-glucosidase polypeptides are provided for delivery to cells or tissues via mRNA therapy, either after formulation of the polyribonucleotide sequence in encapsulated delivery such as liposomes, or as lipid nanoparticles (see, for example, Hou et al., Nature Reviews Materials, 2021, 6:1078-1094).

[0259] In some additional embodiments, the engineered acid alpha-glucosidase polypeptide is provided for delivery to cells or tissues via cell therapy, and the polynucleotide sequence encoding the engineered acid alpha-glucosidase polypeptide is introduced into exogenous cells, which (or more cells) are introduced into a recipient (e.g., a patient with or at risk of developing Pompe disease). Exemplary cells that may be used include, in particular, hematopoietic (blood-forming), stem cells (HSCs), skeletal muscle stem cells, and mesenchymal stem cells.

[0260] Usage and Method In a further embodiment, the disclosure provides the use of engineered acid alpha-glucosidase polypeptides, recombinant polynucleotides encoding engineered acid alpha-glucosidase, or compositions thereof for treating subjects having acid alpha-glucosidase activity deficiency. In some embodiments, engineered acid alpha-glucosidase polypeptides, recombinant polynucleotides, or compositions thereof are used to treat one or more symptoms associated with acid alpha-glucosidase activity deficiency. In some embodiments, engineered acid alpha-glucosidase polypeptides, recombinant polynucleotides, or compositions thereof are used to treat subjects having Pompe disease.

[0261] In some embodiments, a method for treating and / or preventing symptoms of acid alpha-glucosidase activity deficiency comprises providing an effective amount of the engineered acid alpha-glucosidase disclosed herein to a subject in need. In some embodiments, an effective amount of the engineered acid alpha-glucosidase or a pharmaceutical composition thereof is administered to the subject. In some embodiments, the engineered acid alpha-glucosidase is administered at a dose of about 1 mg / kg to about 100 mg / kg. In some embodiments, the engineered acid alpha-glucosidase is administered at a dose of about 10 mg / kg to about 60 mg / kg. In some embodiments, the engineered acid alpha-glucosidase is administered at a dose of about 20 mg / kg to about 40 mg / kg.

[0262] In some embodiments, recombinant polynucleotides encoding engineered acid alpha-glucosidase, or pharmaceutical compositions thereof, are administered to the target. In some embodiments, recombinant polynucleotides encoding engineered acid alpha-glucosidase, or pharmaceutical compositions thereof, are administered in a therapeutically effective dose. In some embodiments, recombinant polynucleotides encoding engineered acid alpha-glucosidase, or pharmaceutical compositions thereof, are administered intravenously, intramuscularly, subcutaneously, intranasally, intraocularly, intrathecally, by inhalation, or orally.

[0263] In some embodiments, the subject of treatment is suffering from Pompe disease. In some embodiments, the symptoms of Pompe disease are alleviated. In some embodiments, the subject of treatment is an infant or child. In some embodiments, the subject of treatment is an adult or adolescent.

[0264] In some embodiments, the disclosure also provides the use of engineered acid alpha-glucosidase in the preparation of a pharmacopoeci for treating acid alpha-glucosidase deficiency in a subject. In some embodiments, the disclosure provides the use of recombinant polynucleotides encoding engineered acid alpha-glucosidase in the preparation of a pharmacopoeci for treating acid alpha-glucosidase activity deficiency in a subject. In some embodiments, the condition in which acid alpha-glucosidase deficiency is involved is Pompe disease.

[0265] The foregoing and other aspects of the present invention may be better understood in relation to the following non-limiting embodiments. These embodiments are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. [Examples]

[0266] In the following experimental disclosures, the following abbreviations apply: ppm (parts per million); M (molar concentration); mM (millimolecular concentration), uM and μM (micromolar concentration); nM (nanomolar concentration); mol (moles); gm and g (grams); mg (milligrams); ug and μg (micrograms); L and l (liters); ml and mL (milliliters); ul, ml, uL, mL (microliters); cm (centimeters); mm (millimeters); um and μm (micrometers); sec. (seconds); min(s) (minutes); h(s) and hr(s) (hours); U (units); MW (molecular weight); rpm (revolutions per minute); °C (degrees Celsius); CDS (code sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coli W3110 (commonly used experimental E. coli strain, Coli Genetic Stock Center [CGSC], New Available from Haven, CT); DPBS (Dulbeccio phosphate-buffered saline); LB (Luria-Bertani); TB (Terrific broth); 4-MUGlu or 4-MU-GLU (4-methylumbelliferyl α-D-glucopyranoside); SD-Ura (uracil-free single dropout medium); HPLC (high-performance liquid chromatography); SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis); MU-Glu (4-methylumbelliferyl α-D-glucopyranoside); IPTG (isopropyl β-D-1-thiogalactopyranoside); PMBS (polymyxin B sulfate); FIOPC (improvement factor against positive control); PBMC (peripheral blood mononuclear cells); LB (Luria broth); and MeOH (methanol).

[0267] Example 1 Obtaining the GAA gene and constructing an expression vector. This example describes the acquisition of the GAA gene and the construction of an expression vector. A synthetic gene encoding WT human GAA (Uniprot ID P10253) was designed for optimized gene expression in Homo sapiens (SEQ ID NO: 1) and cloned into the expression vector pDH (see International Patent Publication No. 2021 / 127457). For secretory expression and transient transfection in mammalian cells, chimeric GAA expression constructs encoding synthetic mouse IG signal peptides fused to synthetic genes encoding different GAA variants (residues 1-19 of Uniprot accession number: A0N1R5) were generated as follows: Fragments encoding the synthetic mouse IG signal peptide and sequences encoding the mature form of GAA were amplified using oligonucleotides containing restriction enzyme neighbors that allowed cloning to either the BamHI / XhoI or HindIII / XhoI site. Acid alpha-glucosidase variants SEQ ID NOs: 3, 7, 5, 13, and 9 were cloned into pDH or pcDNA3.1(+). Using directed evolution, we generated specific gene variants derived from Sequence ID No. 11 (disclosed as Sequence ID No. 3103 in WO2021 / 127457 for polynucleotide sequences and as Sequence ID No. 3104 for polypeptide sequences) within pDH plasmid constructs (see, e.g., U.S. Patent No. 8,383,346 and WO2010 / 144103).

[0268] Example 2 GAA assay obtained by high-throughput growth of suspended mammalian cells and expression of suspended mammalian cells High-throughput (HTP) growth of GAA and GAA variants in suspended mammalian cells (Expi293F) EXPI293F® cells (ThermoFisher Scientific) were transfected with polynucleotides encoding wild-type GAA or GAA variants with synthetic mouse IG signal peptide fusions using lipofection with EXPIFECTAMINE® 293 Reagent (ThermoFisher Scientific) in EXPI293® expression medium (ThermoFisher Scientific). EXPI293F® cells (ThermoFisher Scientific) were cultured in EXPI293® expression medium (ThermoFisher Scientific) and transferred to 1.1 mL deep-well plates (Corning, P-DW-11-CS) of Axygen, with 1 × 10⁶ cells per plate. 6 Cells were seeded at a density of 400 μL per well. Lipofection-mediated transfection was performed on the cells, and they were returned to a shaking incubator at 8% CO2 and 70% humidity for 3–4 days to allow expression of the GAA variant and secretion into the conditioned medium. The expression plates were centrifuged, and the conditioned medium was recovered by transferring it to a BioRad Hardshell PCR Plate (BioRad, HSP9601). The plates were centrifuged again for clarification, and the conditioned medium was transferred to a new 96-well plate for activity, stability, or incorporation into cell analysis.

[0269] HTP analysis of the supernatant GAA variant activity was determined by measuring the hydrolysis of 4-methylumbelliferyl α-D-glucopyranoside (4-MU-GLU). For the unchallenged assay, 5 μL of EXPI293F™ clarified and conditioned medium, prepared as described above, was mixed with 50 μL of 1.5 mM 4-MU-GLU in McIlvaine buffer (McIlvaine, J. Biol. Chem., 1921, 49:183-186), pH 4.4, in a 96-well black opaque bottom plate. The reaction mixture was incubated at 25°C for 15 minutes with stirring at 400 rpm, and then quenched with 100 μL of 0.5 M sodium carbonate (pH 10.5). Hydrolysis was analyzed using an EnVision microplate reader (Perkin Elmer) monitoring fluorescence (excitation 355 nm, emission 460 nm). Unchallenged activity FIOPC was calculated by dividing the normalized GAA variant by the activity of the reference polypeptide having the indicated sequence number.

[0270] HTP analysis of supernatant obtained from a challenge test using plasma To simulate the conditions under which the variant would encounter in the blood after administration to a patient, the GAA variant was challenged in plasma. First, 30 μL of the GAA variant in EXPI293F™ clarified and conditioned medium was combined with 30 μL of plasma (Innovative Research, Innovative Grade US Origin Monkey Cynomolgus Plasma K2 EDTA) in a 96-well plate. The plate was sealed and incubated at 37°C for 4 hours with agitation at 400 rpm. Next, 10 μL of the plasma challenged sample was mixed with 50 μL of 1.5 mM 4-MU-GLU in McIlvaine buffer at pH 4.4. The reaction was incubated at 25–37°C for 30 minutes with agitation at 400 rpm, and then quenched with 100 μL of 0.5 M sodium carbonate (pH 10.5). Hydrolysis was analyzed using an EnVision microplate reader (Perkin Elmer) that monitors fluorescence (excitation at 355 nm, emission at 460 nm). Plasma stability FIOPC was calculated by dividing the normalized GAA variant activity after the challenge trial by the activity of the reference polypeptide with the indicated SEQ ID NO after the challenge trial.

[0271] HTP analysis of GAA activity in lysates of Pompe fibroblasts GAA variants derived from HTP EXPI293F® expression in clarified and conditioned medium were incubated with target cells, and residual intracellular activity was assayed after 72 hours. For these experiments, mammalian cells lacking functional GAA activity, namely Pompe patient-derived fibroblasts (Coriell Institute for Medical Research, no. GM00248), were used. Pompe patient-derived fibroblasts were seeded in 96-well COSTAR® plates (3904, Corning) and grown in standard full growth medium until confluence. Once confluence was reached, the full growth medium was removed from the plate using an automated BioMek i5 liquid handler. Clarified and conditioned medium from transient HPT transfection in EXPI293F® was transferred to Pompe patient-derived fibroblasts and incubated at 37°C and 5% CO2 for 24 hours. The medium was removed from the culture using an automated BioMek i5 liquid handler. Cells were briefly washed with 150 μL of 1× DPBS / well, and the DPBS was removed using an automated BioMek i5 liquid handler. Then, 200 μL of standard full-growth culture medium was added to each well, and the plate was returned to the incubator for 72 hours. At the end of incubation, the standard full-growth medium was removed using an automated BioMek i5 liquid handler. Cells were washed with 150 μL of 1× DPBS / well, and the DPBS was removed using an automated BioMek i5 liquid handler. Cells were lysed by adding 50 μL of McIlvaine buffer (pH 4.4), supplementing with 0.5% TRITON® X-100® nonionic surfactant (Sigma, catalog no. 93443), and stirring at room temperature for 30 minutes. Activity was evaluated by adding 50 μL of 1.5 mM 4-MU-GLU to McIlvaine buffer at pH 4.4. The plate was sealed and incubated at 37°C for 360 minutes with stirring at 400 rpm, then quenched with 100 μL of 0.5 M sodium carbonate (pH 10.5).Hydrolysis was analyzed using an EnVision microplate reader (Perkin Elmer) that monitors fluorescence (excitation 355 nm, emission 460 nm). Cellular uptake FIOPC was calculated by dividing the intracellular activity of the normalized GAA variant by the activity of the reference polypeptide having the indicated SEQ ID NO.

[0272] Example 3 GAA variant of SEQ ID NO. 11 In this example, experiments for the evolution and screening of GAA variants derived from SEQ ID NO. 11 for improved GAA activity after a series of challenge tests are described. A library of variant genes GAA encoded based on SEQ ID NO. 11 was constructed, plated, grown, and screened for GAA 4-MU-GLU activity (“unchallenged activity FIOPC”), and after a plasma challenge test (“plasma stability and activity FIOPC”). The variants were also tested for 4-MU-GLU activity after lysis of Pompe fibroblast cells treated with conditioned medium as described in Example 2 (“lysate activity FIOPC from treatment of Pompe fibroblast cells”). The results of these assays are shown in Table 3-1.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

[0273] Example 4 Production of GAA variants GAA production in EXPI293F(trademark) cells Milligram-scale production of the GAA variant was achieved by transient transfection of EXPI293F® cells (ThermoFisher Scientific) using a lipofection method with EXPIFECTAMINE® 293 reagent (ThermoFisher Scientific) in EXPI293® expression medium (ThermoFisher Scientific). The GAA variant fused to the N-terminal synthetic mammalian signal peptide was subcloned into the mammalian expression vector pDH or pcDNA 3.1(+) as described in Example 1. EXPI293F® cells were transfected with plasmid DNA and grown in suspension for 4–7 days. The conditioned medium was then collected, clarified by centrifugation and filtration or by diatomaceous earth, and stored at -80°C until purified.

[0274] Example 5 Purification of GAA variants As described in Example 4, GAA variants (SEQ ID NOs: 2, 4, 12, 8, 6, 14, 10) produced by EXPI293F® cells were purified from mammalian culture supernatant as described in the literature (Yasuda et al., Prot.Exp.Pur., 2004, 37:499-506). Concanavalin A resin (Sigma Aldrich) was equilibrated with 0.1 M sodium acetate, 0.1 M NaCl, 1 mM MgCl2, CaCl2, and MnCl2, pH 6.0 (concanavalin A binding buffer). The supernatant was concentrated 10-fold and diluted 2-fold to equilibration buffer (0.1 M sodium acetate, 0.1 M NaCl, 1 mM MgCl2, CaCl2, and MnCl2, pH 6.0), and then packed into a column. After packing, the column was washed with 10 column volumes of concanavalin A-binding buffer, and the bound protein was eluted with concanavalin A-binding buffer supplemented with 0.9 M methyl-α-D-mannopyranoside and 0.9 M methyl-α-D-glucopyranoside. The eluted protein was concentrated, and the buffer was replaced with storage buffer (20 mM sodium phosphate, 150 mM sodium chloride, 185 μM TWEEN®-20 nonionic detergent, pH 6.0) using an AMICON® Ultra 15 mL filtration unit with a 50 kDa molecular weight cutoff membrane (Millipore). The GAA in the storage buffer was aseptically filtered through an ANOTOP® 0.2 μm syringe filter (Whatman) and stored at -80°C. The purification process produced 60–100 mg of purified protein / culture supernatant (L).

[0275] Example 6 In vitro characteristic determination of GAA variant This embodiment describes experiments performed to characterize the GAA variant produced as shown herein.

[0276] Stability of rhGAA and GAA variants at neutral pH The stability of the GAA variant to neutral pH was determined by incubating the purified variant at 214 nM in MEM full growth medium (pH 7.4) in a 96-well plate. The plate was incubated at 37°C for up to 144 hours. At each time point, 10 μL of the neutral pH challenged sample was transferred to a BioRad hardshell plate and immediately frozen at -80°C. At the end of the experiment, all plates were treated simultaneously by adding 50 μL of 1.5 mM 4-MU-GLU to McIlvaine buffer for 30 minutes with stirring at 400 rpm at 37°C. The reactants were quenched with 100 μL of Na2CO3 (0.5 M, pH 10.5), 100 μL was transferred to a black 96-well plate, and hydrolysis was evaluated by quantifying the released fluorescent methylumbelliferone using an Envision microplate reader (excitation 355 nm / emission 460 nm). The results from this assay are shown in Figure 1.

[0277] Melting temperatures of rhGAA and GAA variants at neutral pH and lysosomal pH. The melting temperatures of rhGAA and GAA variants were determined by differential scanning fluorescence assay at neutral pH and lysosomal pH. GAA enzyme variants were diluted to 1 mg / mL in DPBS (pH 6.2). 40 μL of McIlvaine buffer at pH 4.4 or pH 7.4 containing 1×SYPRO Orange was mixed with 10 μL of each enzyme solution (n=3) in separate wells of a 96-well skirted BioRad plate. The plates were sealed with optically clear film and run on a CFX connect real-time PCR system using the manufacturer's recommended method at 25–95°C. Data were analyzed using BioRad Software. The results of these assays are shown in Figure 2.

[0278] Plasma stability of rhGAA and GAA variants The stability of GAA variants in cynomolgus monkey plasma was evaluated over a 75-hour time course. Purified GAA variants were diluted in GAA storage buffer (20 mM sodium phosphate, 150 mM NaCl, 185 μM polysorbate 20, pH 6.0) to a concentration of 100 μg / mL (2 × assay concentration) and dispensed into 3-well BioRad hard-shell plates. 100 μL of cynomolgus monkey plasma was added and thoroughly mixed. The plasma challenge plates were sealed and incubated at 37°C with agitation (400 rpm) for a maximum of 75 hours. At the end of the challenge time, the residual activity of the GAA variant was measured by transferring 10 μL of the challenged plasma solution to 50 μL of 1.5 mM 4-MU-GLU in McIlvaine buffer (pH 4.4) in a black 96-well plate over 30 minutes with agitation (400 rpm) at 37°C. Hydrolysis was evaluated by quenching the reaction with 100 μL of Na2CO3 (0.5 M, pH 10.5) and quantifying the released fluorescent methylumbelliferone using an Envision microplate reader (excitation 355 nm / emission 460 nm). The results from this assay are shown in Figure 3.

[0279] Cell uptake of purified GAA variants in Pompe fibroblasts or C2C12 GAA knockout myoblasts The ability of GAA variants to cross-correct cells compared to reference enzymes (SEQ ID NOs: 2 and 4) was determined. Pompe fibroblasts (GM00248, Coriell Institute for Medical Research) or C2C12 GAA knockout myoblasts were seeded in standard full-growth medium in black-walled, clear-bottomed 96-well plates (Costar, No. 3604) and allowed to reach confluence (2–3 days at 37°C, 5% CO2). After reaching confluence, the standard full-growth medium was removed using an automated BioMek i5 liquid handler. The purified enzyme, as described in Example 5, was added to the cells in standard full-growth medium at a series of dilutions ranging from 0 to 214 nM GAA / mL and incubated at 37°C, 5% CO2 for 1, 4, 24, or 96 hours. At the end of the process, the medium containing the GAA variant was aspirated using an automated BioMek i5 liquid handler. Cells were briefly washed with 150 μL of 1× DPBS / well, and the DPBS was removed using an automated BioMek i5 liquid handler. Then, 200 μL of standard full growth medium was added to each well, and the plate was returned to the incubator for the remainder of the experiment (95-0 hours depending on processing time). At the end of the experiment, the MEM full growth medium was removed using an automated BioMek i5 liquid handler. Cells were washed with 150 μL of 1× DPBS / well, and the DPBS was removed using an automated BioMek i5 liquid handler. Cells were lysed by adding 50 μL of McIlvaine buffer (pH 4.4), supplementing with 0.5% TRITON® X-100® nonionic surfactant (Sigma, no. 93443), and stirring at room temperature for 30 minutes. GAA activity was evaluated by adding 50 μL of 1.5 mM 4-MU-GLU to McIlvaine buffer at pH 4.4. The plates were sealed and incubated at 37°C for 300–360 minutes with stirring at 400 rpm, followed by quenching with 100 μL of 0.5 M sodium carbonate (pH 10.5).Hydrolysis was analyzed using an EnVision (Perkin Elmer) microplate reader that monitors fluorescence (excitation 355 nm, emission 460 nm). Panels A, B, C, and D in Figure 4 provide graphs showing the activity in Pompe fibroblast lysates after treatment with purified GAA variants over a period of 1–96 hours. Panels A, B, C, and D in Figure 5 provide graphs showing the activity of purified GAA variants in C2C12 GAA KO myoblast lysates over a period of 1–96 hours.

[0280] Example 7 In vitro characterization of GAA variant expression in myoblasts This example describes experiments performed to characterize the expression and activity of GAA variants in myoblasts as described herein.

[0281] rhGAA, GAA variant 4-MU-GLU, and glycogen hydrolysis activity in transiently transfected myoblasts Transient transfection was used to evaluate the expression efficiency and activity of wild-type GAA and GAA variants in myoblasts. C2C12 GAA knockout myoblasts were seeded in 12-well plates in full growth medium (Dulbecc's modified Eagle medium containing 10% fetal bovine serum) and allowed to adhere for 24 hours until approximately 50% confluence. Cells were transfected with GAA variant plasmid DNA using jetOPTIMUS® transfection polyplus. The plates were returned to the incubator for 4 hours before changing the medium. On day 3 post-transfection, the acclimatization medium was evaluated for GAA activity, and myoblasts were harvested to evaluate GAA activity in the lysates. GAA activity in conditioned medium was determined relative to 4-MU-GLU and evaluated by incubation of 20 μL of conditioned medium containing 50 μL of 1.5 mM 4-MU-GLU in McIlvaine buffer (pH 4.4) in a black 96-well plate with stirring (400 rpm) at 37°C. Hydrolysis was evaluated by quantifying the released fluorescent methylumbelliferone using an Envision microplate reader (excitation 355 nm / emission 460 nm). To produce lysates, myoblasts were harvested with trypsin and centrifuged. The cell pellet was washed with DPBS and intermittently vortexed on ice for 30 minutes in 52 μL of GAA lysis buffer (0.2 M sodium acetate, 0.4 M potassium chloride, 0.5% Triton® X-100, pH 4.3). The lysate was clarified (at 20,000 RCF for 10 minutes), and the protein concentration was determined and normalized by BCA. The GAA activity in the lysate relative to 4-MU-GLU was evaluated by incubation of 2 μL or 4 μL of normalized lysate containing 50 μL of 1.5 mM 4-MU-GLU in McIlvaine buffer (pH 4.4) for 4 hours in a black 96-well plate with stirring (400 rpm) at 37°C. Hydrolysis was evaluated by quantifying the released fluorescent methylumbelliferone using an Envision microplate reader (excitation 355 nm / emission 460 nm).The glycogen hydrolysis activity of the lysate was determined by incubating the lysate in glycogen (100 mg / ml prepared in GAA reaction buffer of 0.1 M sodium acetate, 0.1 M NaCl, and 0.5 mg / mL BSA at pH 4.3) for 1 hour at 37°C and 400 rpm. The glycogen hydrolysis reaction was quenched / neutralized by adding 90 μL of stop buffer (133 mM glycine, 83 mM sodium carbonate, pH 10.7). The quenched hydrolysis reaction was diluted 1:20 in Amplex Red reaction buffer (Invitrogen Amplex Red Glucose / Glucose Oxidase Assay Kit No. A22189). 50 μL of the quenched hydrolysis reaction dilution and 50 μL of glucose standard solutions (0, 3.2, 6.25, 12.5, 25, 50, and 100 μM) were added to a black 96-well plate. 50 μL of Amplex Reagent Mix (Amplex Red / HRP / glucose oxidase) was added to all wells, and the plates were incubated at room temperature with gentle shaking and protected from light for 30 minutes. Red fluorescent resolfins (formed from the reaction of Amplex Red with hydrogen peroxide produced from the glucose oxidase-HRP binding reaction) were quantified using a Spectramax EM microplate reader (excitation 540 nm / emission 590 nm). Figure 6 provides a graph showing the activity of C2C12 GAA KO myoblasts in conditioned medium after transient transfection. Figure 7 provides graphs (panels A and B) showing the activity of C2C12 GAA KO myoblasts in lysates after transient transfection.

[0282] Example 8 Identification of active GAA variants with reduced immunogenicity This example describes experiments performed to characterize the expression and activity of GAA variants in myoblasts as described herein.

[0283] The putative T cell epitopes in WT GAA (SEQ ID NO: 2) or the engineered GAA variant (SEQ ID NO: 12) were identified using the Immune Epitope Database (IEDB; Immune Epitope Database and Analysis Resource website), a tool known in the art, as well as proprietary statistical analysis tools (e.g., iedb.org, and Vita et al., Nucl. Acids Res., 2020, 38 (Database issue): D854-62. Epub 2009 Nov 11). WT GAA or the engineered GAA variant was analyzed into all possible 15-mar analysis frames, with each frame overlapping with the last 14 amino acids. The immunogenicity of 15 MAR analysis frames was assessed by scoring their predicted binding to eight common class II HLA-DR alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, and DRB1*1501) that collectively cover 77% of the world's population, using the method recommended on the IEDB website (see, e.g., iedb.org and Bui et al., 2006, BMC Bioinformatics, 7:153). Potential T cell epitope clusters contained within the enzyme (i.e., subregions contained within GAA with an unusually high potential for immunogenicity) were identified using statistical analysis tools, as is known in the art.

[0284] In Example 3, the GAA variant identified as active in the assay described in Example 2 was analyzed for its predicted level of immunogenicity by evaluating its binding to eight common class II HLA-DR alleles. For each variant, the total immunogenicity score and immunogenicity hit count were calculated. The total immunogenicity score (TIS) reflects the total number of predicted binding events between the eight MHC class II alleles (described above) and peptide 15 mers across the entire sequence (i.e., a higher score indicates a higher level of predicted immunogenicity). The immunogenicity "hit count" (IHC) indicates the number of peptide 15 mers predicted to bind to four or more of the eight common alleles. These regions of the sequence are particularly likely to be immunogenic across the population (i.e., a higher score indicates a higher likelihood of immunogenicity). Mutations that cause a reduced total immunogenicity score and / or immunogenicity hit count compared to the reference sequence are considered potential "immunoeliction mutations" and are shown in Table 4-1. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10]

[0285] Example 9 Characterization of GAA variants in ex vivo immunogenicity assessment MHC II-related peptide proteomics (MAPPs) assays can provide experimental evidence of HLA-binding epitopes from protein antigens. MAPP assays combine the key steps of antigen uptake into differentiated antigen-presenting cells, lysosomal processing, HLA binding, and presentation on the cell surface of antigen-presenting cells. The naturally processed, bound, and presented peptides are then identified and quantified by liquid chromatography-mass spectrometry. Presentation of peptides on the cell surface by HLA-DR receptors to CD4+ T helper cells is a necessary step in the activation cascade, including T cell proliferation, differentiation, and the eventual production of antibodies from B cells. Therefore, reduced binding of processed antigens to the grooves of MHC II molecules, and subsequent reduced antigen presentation, are thought to reduce the potential immunogenicity risk of the antigen.

[0286] In this example, peripheral blood mononuclear cells (PBMCs) were isolated from a healthy donor buffy coat, and CD14+ monocytes were isolated and subsequently differentiated into dendritic cells (DCs) by methods known in the art. The DCs were then incubated with GAA variants and induced into a mature phenotype by adding lipopolysaccharide. HLA-DR binding peptides resulting from the treated antigens (GAA and variants) were captured by immunoprecipitation and eluted for LC-MS analysis. The identified binding peptides were aligned with the amino acid sequence of the GAA variants to allow for comparison of the treatment and abundance of peptides from each GAA variant (Kropshofer and Spindeldreher (2005) in Antigen Presenting Cells: From Mechanisms to Drug Development, eds. Kropshofer and Vogt, Wiley-VCH, Weinheim, 159-98). By comparing the identified peptides, the GAA variants of SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 14 experimentally showed significantly reduced treatment and reduced peptide presentation frequency compared to SEQ ID NO: 2 (see Figure 8).

[0287] Although the present invention has been described with reference to specific embodiments, various modifications can be made, and equivalents can be substituted to adapt to specific situations, materials, substance compositions, processes, process steps, or steps, thereby achieving the benefits of the present invention without departing from the claims.

[0288] All publications, patents, patent applications, and other documents cited herein are incorporated herein by reference in whole for all purposes to the same extent as each individual publication, patent, patent application, or other document is individually indicated to be incorporated by reference for all purposes.

Claims

1. An engineered acid alpha-glucosidase, or a biologically active fragment thereof, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity with respect to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12, or to a reference sequence corresponding to SEQ ID NO: 12, wherein the amino acid sequence comprises at least substitutions or amino acid residues 305V, 24A / C / D / F / G / H / I / K / M / N / P / S / T / V / Y, 28A / C / D / E / F / G / H / K / Q / T / V / W, 29A / C / D / E / F / G / H / I / K / M / N / P / R / W / Y, 39A / E / F / G / I / L / N / T, 50A / C / D / E / F / H / I / K / M / N / R / S / T / W / Y, 62D / H / I / K / M / N / P / Q / Y, 78A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 8 7A / G / H / I / K / L / MN / Q / R / S / T / V / W, 135C / D / E / F / G / H / I / K / L / N / R / Y, 266A / D / E / H / K / Q, 267H / L / T / V, 437A / H, 486C / D / F / G / H / I / K / L / M / N / Q / R / S / V / W / Y, 52 2A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y, 569A / C / D / E / G / K / M / N / P / R / W, 6 70A / D / G / H / K / M / Y, 692A / D / E / H / K / L / M / N / T / W, 711D / E / I / K / M / N / Q / S / T / V / Y, 736F / L, 750E / K / L / Q / R, 812A / D / G / S, 830D / E / F / G / H / L / M / N / S / T / W / Y, 842 A / C / D / F / H / K / L / M / N / Q / R / T / W, 871A / C / D / F / H / I / M / N / Q / T / V / W / Y, 883A / F / Q, 894A / D / E / H / I / K / L / M / N / S / T / V / W / Y, 913F / I / K / M / N / S, or 932C / D / E / G / H / K / L / M / N / P / Q / R / W / Y, or combinations thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 20-944 of SEQ ID NO: 12 or 2, or to a reference sequence corresponding to SEQ ID NO: 12 or 2, manipulated acid alpha-glucosidase,or its biologically active fragment.

2. An engineered acid alpha-glucosidase, or a biologically active fragment thereof, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity with respect to a reference sequence corresponding to residues 20–944 of even-numbered sequence codes 14–754, or with respect to even-numbered sequence codes 14–754, wherein the amino acid sequence comprises at least a substitution or amino acid residue 305V , 24A / C / D / F / G / H / I / K / M / N / P / S / T / V / Y, 28A / C / D / E / F / G / H / K / Q / T / V / W, 29A / C / D / E / F / G / H / I / K / M / N / P / R / W / Y, 39A / E / F / G / I / L / N / T, 50A / C / D / E / F / H / I / K / M / N / R / S / T / W / Y, 62D / H / I / K / M / N / P / Q / Y, 78A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 87A / G / H / I / K / L / MN / Q / R / S / T / V / W, 135C / D / E / F / G / H / I / K / L / N / R / Y, 266A / D / E / H / K / Q, 267H / L / T / V, 437A / H, 486C / D / F / G / H / I / K / L / M / N / Q / R / S / V / W / Y, 522A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y, 569A / C / D / E / G / K / M / N / P / R / W, 670A / D / G / H / K / M / Y, 692A / D / E / H / K / L / M / N / T / W, 711D / E / I / K / M / N / Q / S / T / V / Y, 736F / L, 750E / K / L / Q / R, 812A / D / G / S, 830D / E / F / G / H / L / M / Manipulated acid alpha-glucosidases comprising N / S / T / W / Y, 842A / C / D / F / H / K / L / M / N / Q / R / T / W, 871A / C / D / F / H / I / M / N / Q / T / V / W / Y, 883A / F / Q, 894A / D / E / H / I / K / L / M / N / S / T / V / W / Y, 913F / I / K / M / N / S, or 932C / D / E / G / H / K / L / M / N / P / Q / R / W / Y, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 20-944 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2.or its biologically active fragment.

3. The manipulated acid alpha-glucosidase according to claim 1 or 2, wherein the amino acid sequence of the manipulated acid alpha-glucosidase includes residues 20 to 944 of even-numbered sequence numbers 14 to 754, or includes even-numbered sequence numbers 14 to 754.

4. The manipulated acid alpha-glucosidase according to claim 1 or 2, wherein the amino acid sequence of the manipulated acid alpha-glucosidase comprises residues 20 to 944 of SEQ ID NO: 14, 114, 126, 170, 250, 252, 394, 472, 488, or 506, or comprises SEQ ID NO: 14, 114, 126, 170, 250, 252, 394, 472, 488, or 506.

5. The manipulated acid alpha-glucosidase according to any one of claims 1 to 4, wherein the manipulated acid alpha-glucosidase exhibits, compared to a reference acid alpha-glucosidase having a sequence corresponding to residues 20 to 944 of SEQ ID NO: 2 or 12, or a sequence corresponding to SEQ ID NO: 2 or 12, at least one improved property selected from any combination of i) ii) iii) iv) v) vi) vii) viiii) and ix).

6. The manipulated acid alpha-glucosidase according to claim 5, wherein the manipulated acid alpha-glucosidase exhibits reduced immunogenicity compared to a reference acid alpha-glucosidase having a sequence corresponding to residues 20-944 of SEQ ID NO: 2 or 12, or a sequence corresponding to SEQ ID NO: 2 or 12.

7. The manipulated acid alpha-glucosidase according to claim 6, wherein the manipulated acid alpha-glucosidase exhibits (a) a decrease in total immunogenicity score (TIS) of more than 10 compared to the reference acid alpha-glucosidase of SEQ ID NO: 2, (b) a decrease in immunogenicity hit count (IHC) of more than 2 compared to the reference acid alpha-glucosidase of SEQ ID NO: 2, (c) a decrease in total immunogenicity score (TIS) of more than 10 compared to the reference acid alpha-glucosidase of SEQ ID NO: 12, and / or (d) a decrease in immunogenicity hit count (IHC) of more than 2 compared to the reference acid alpha-glucosidase of SEQ ID NO:

12.

8. The manipulated acid alpha-glucosidase according to any one of claims 1 to 7, comprising the manipulated acid alpha-glucosidase prepropeptide.

9. The manipulated acid alpha-glucosidase according to claim 8, wherein the prepropeptide of the manipulated acid alpha-glucosidase comprises a eukaryotic or synthetic signal peptide sequence.

10. The manipulated acid alpha-glucosidase according to claim 9, wherein the signal peptide comprises a mouse or human signal peptide sequence.

11. The manipulated acid alpha-glucosidase according to any one of claims 1 to 7, comprising the propeptide of the manipulated acid alpha-glucosidase.

12. The manipulated acid alpha-glucosidase according to any one of claims 1 to 11, wherein the manipulated acid alpha-glucosidase is purified.

13. A pharmaceutical composition comprising the manipulated acid alpha-glucosidase described in any one of claims 1 to 12.

14. The pharmaceutical composition according to claim 13, further comprising a pharmaceutically acceptable carrier and / or excipient.

15. The pharmaceutical composition according to any one of claims 13 or 14, wherein the composition is suitable for parenteral injection or infusion into humans.

16. A recombinant polynucleotide comprising a polynucleotide sequence encoding the manipulated acid alpha-glucosidase described in any one of claims 1 to 11.

17. The recombinant polynucleotide according to claim 16, comprising a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity with respect to a reference polynucleotide sequence corresponding to nucleotide residues 58 to 2832 of the odd-numbered sequence numbers of sequence numbers 13 to 753, wherein the polynucleotide encodes an acid alpha-glucosidase.

18. The recombinant polynucleotide according to claim 16 or 17, comprising a codon-optimized polynucleotide sequence for the expression of the encoded manipulated acid alpha-glucosidase.

19. The recombinant polynucleotide according to claim 16, comprising a polynucleotide sequence containing nucleotide residues 58 to 2832 of odd-numbered sequence numbers 13 to 753, or a polynucleotide sequence containing odd-numbered sequence numbers 13 to 753.

20. The recombinant polynucleotide according to claim 16, comprising a polynucleotide sequence containing nucleotide residues 58 to 2832 of sequence numbers 13, 113, 125, 169, 249, 251, 393, 471, 487, or 505, or a polynucleotide sequence containing sequence numbers 13, 113, 125, 169, 249, 251, 393, 471, 487, or 505.

21. An expression vector comprising a recombinant polynucleotide according to any one of claims 16 to 20.

22. The expression vector according to claim 21, wherein the recombinant polynucleotide is operably linked to a control sequence.

23. The expression vector according to claim 22, wherein the control sequence includes a promoter.

24. The expression vector according to claim 23, wherein the promoter is a heterologous promoter.

25. A host cell comprising the expression vector according to any one of claims 21 to 24.

26. The host cell according to claim 25, wherein the host cell is a eukaryotic cell or a prokaryotic cell.

27. The host cell according to claim 25, wherein the host cell is a mammalian cell.

28. The host cell according to claim 27, wherein the mammalian cell is a human cell.

29. The host cell according to claim 28, wherein the human cell is derived from a patient having a deficiency in acid alpha-glucosidase activity.

30. A method for producing an engineered acid alpha-glucosidase variant, comprising culturing a host cell according to any one of claims 25 to 29 under conditions that produce the acid alpha-glucosidase encoded by the recombinant polynucleotide.

31. The method according to claim 30, further comprising the step of recovering the acid alpha-glucosidase.

32. The method according to claim 30 or 31, further comprising the step of purifying the acid alpha-glucosidase.

33. A method for treating and / or preventing symptoms of acid alpha-glucosidase deficiency in a subject, comprising administering to a subject in need an effective amount of the manipulated acid alpha-glucosidase described in any one of claims 1 to 12, or the pharmaceutical composition described in any one of claims 13 to 15.

34. The method according to claim 33, wherein the deficiency of acid alpha-glucosidase is Pompe disease.

35. The method according to any one of claims 33 or 34, wherein the subject is an infant or a child.

36. The method according to any one of claims 33 or 34, wherein the subject is an adult or a minor.

37. Use of the manipulated acid alpha-glucosidase according to any one of claims 1 to 12 for the treatment of acid alpha-glucosidase deficiency.

38. Use of the manipulated acid alpha-glucosidase according to any one of claims 1 to 12 in the preparation of a pharmaceutical for treating a subject having an acid alpha-glucosidase deficiency.

39. The use according to claim 37 or 38, wherein the deficiency of acid alpha-glucosidase is Pompe disease.

Images