Manipulated acidic alpha-glucosidase variant

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Engineered GAA polypeptides with improved expression, stability, and cellular uptake enhance enzyme activity, addressing the limitations of current therapies for Pompe disease and reducing glycogen accumulation.

JP7865532B2Active Publication Date: 2026-05-26CODEXIS INC

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Patent Information

Authority / Receiving Office: JP · JP
Patent Type: Patents
Current Assignee / Owner: CODEXIS INC
Filing Date: 2020-12-18
Publication Date: 2026-05-26

Application Information

Patent Timeline

18 Dec 2020

Application

26 May 2026

Publication

JP7865532B2

IPC: C12N9/26; C12N15/56; C12N15/63; C12N1/15; C12N1/19; C12N1/21; C12N5/10; A61K38/47; A61K48/00; A61P3/00; A61P43/00

CPC: A61K38/00; C12N9/2428; C12Y302/0102; Y02E50/10; C12Y302/01003; C12N9/2402; A61K38/47; A61P21/00

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AI Technical Summary

Technical Problem

Current enzyme replacement therapies for Pompe disease, such as recombinant GAA, do not adequately address the wide range of phenotypes and severity of the disease, particularly in infantile and late-onset forms, due to insufficient enzyme activity and stability, leading to severe cellular defects and organ dysfunction.

Method used

Engineered acid alpha-glucosidase (GAA) polypeptides with optimized expression, stability at neutral and acidic pH, and increased cellular uptake, along with enhanced activity in cell lysates, are developed to improve therapeutic efficacy.

Benefits of technology

The engineered GAA polypeptides provide increased enzyme activity and stability, addressing the limitations of existing therapies and potentially improving patient outcomes by reducing glycogen accumulation and associated cellular damage.

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Abstract

The present invention provides engineered acid alpha-glucosidase (GAA) polypeptides and compositions thereof. In some embodiments, the engineered GAA polypeptides are optimized to provide increased expression, increased stability at neutral pH, and increased activity in cell lysates. The present invention also provides methods for utilizing compositions comprising the engineered GAA polypeptides for therapeutic and other purposes.

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Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 951,625, filed on 20 December 2019, which is thus incorporated herein by reference in its entirety for all purposes.

[0002] Field of Invention This invention provides engineered acid alpha-glucosidase (GAA) polypeptides and compositions thereof. In some embodiments, the engineered GAA polypeptides are optimized to provide increased expression, increased stability at neutral and acidic pH, increased uptake into cells, and increased activity in cell lysates. This invention also provides methods for utilizing compositions comprising engineered GAA polypeptides for therapeutic and other purposes.

[0003] References to sequence listings, tables, or computer programs An official copy of the sequence listing is filed via EFS-Web at the same time as this specification, as an ASCII text file named "CX7-199WO2_ST25.txt", created on December 17, 2020, and measuring 18.2 megabytes. This sequence listing, filed via EFS-Web, is part of this specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] Background of the Invention Pompe disease is an autosomal recessive lysosomal storage disorder caused by a mutation in the gene encoding acid alpha-glucosidase. This genetic defect leads to a reduction or absence of GAA in body tissues. The resulting accumulation of glycogen in lysosomes can lead to lysosomal swelling and rupture, resulting in cytotoxicity, organelle dysfunction, and other cellular defects. There are two types of Pompe disease, including the classic infantile type and the late-onset (childhood or adult) type. Disease severity is related to the amount of enzyme activity present in the cells of the affected individual. The infantile type is the most severe and rapidly progressive type, usually with GAA activity of less than 1%, resulting in significant glycogen accumulation in skeletal muscle as well as the heart and other tissues (see, for example, Hahn and Schanzer, Ann. Transl. Med., 7:283

[2019] ). These patients have multisystemic storage of lysosomes and non-lysosomal-bound glycogen accumulated in the heart, skeletal muscle, and brain tissue (see Schoser, Ann. Transl. Med., 7:292

[2019] ). Patients present with elevated creatinine kinase levels, hypertrophic cardiomyopathy, growth retardation, hypotonia, and axial muscle weakness. If left untreated, patients usually die within the first year of life due to cardiopulmonary failure. Survival beyond 18 months of age is exceptional. This type is distinguished from non-classical or late-onset infantile Pompe disease, in which patients present with much less severe cardiac hypertrophy. Patients with late-onset Pompe disease generally experience progressive limb-girdle myopathy and respiratory dysfunction. These patients present with predominant, but not exclusively, muscle lesions. Patients eventually become wheelchair and / or mechanically ventilated. Respiratory failure is the leading cause of death in these patients. Some patients can synthesize non-functional forms of GAA, while others are unable to produce any type of natural enzyme.The human GAA gene encoding GAA has been identified on chromosome 17, q25.2–q25.3, and has been cloned and sequenced (see Peruzzo et al., Ann. Transl. Med., 7:278–287

[2019] ; and Martiniuk et al., DNA Cell. Biol., 10:283–292

[1991] ). Although numerous mutations in this gene have been reported, the pathological mechanisms that lead to the wide range of phenotypes observed in affected patients remain unclear. Despite the availability of enzyme replacement therapy (ERT) using recombinant GAA, better treatment and management options for affected patients are still needed. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Hahn and Schanzer, Ann. Transl. Med., 7:283

[2019] [Non-Patent Document 2] Schoser, Ann. Transl. Med., 7:292

[2019] [Non-Patent Document 3] Peruzzo et all, Ann. Transl. Med., 7:278-287

[2019] [Non-Patent Document 4] Martiniuk et al., DNA Cell. Biol., 10:283-292

[1991] [Overview of the Initiative] [Means for solving the problem]

[0006] Summary of the Invention This invention provides engineered acid alpha-glucosidase (GAA) polypeptides and compositions thereof. In some embodiments, the engineered GAA polypeptides are optimized to provide increased expression, increased stability at neutral and acidic pH, increased uptake into cells, and increased activity in cell lysates. This invention also provides methods for utilizing compositions comprising engineered GAA polypeptides for therapeutic and other purposes. In some embodiments, the present invention provides engineered GAA polypeptides (also referred to herein as “recombinant GAA polypeptides”), as well as their bioactive fragments and analogs, which have improved properties when compared to wild-type GAA enzymes and / or reference GAA polypeptides under essentially the same conditions. The present invention further relates to methods of using engineered GAA polypeptides, as well as their bioactive fragments and analogs, in therapeutic and / or other compositions.

[0007] The present invention provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with respect to SEQ ID NOs. 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104. In some embodiments, the present invention provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104. In some embodiments, the present invention further provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising amino acid sequences consisting of SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104. In some embodiments, the recombinant acid alpha-glucosidase sequence comprises a signal peptide sequence encoded by polynucleotides represented by SEQ ID NOs: 3381 and 3383, respectively (e.g., SEQ ID NOs: 3382 or 3384).In some embodiments, the recombinant polynucleotide encoding the recombinant acid alpha-glucosidase of the present invention includes a 57-base pair sequence encoding a signal peptide. In some embodiments, the polypeptide of the recombinant acid alpha-glucosidase of the present invention includes a 19-amino acid signal peptide. In some alternative embodiments, the recombinant polynucleotide encoding the recombinant acid alpha-glucosidase does not include a sequence encoding a signal peptide. In some additional embodiments, the recombinant polypeptide containing recombinant acid alpha-glucosidase does not include a signal peptide. The present invention is not intended to be limited to recombinant acid alpha-glucosidase polynucleotides or polypeptide sequences that include a signal peptide nucleotide or polypeptide sequence. The present invention is also not intended to be limited to recombinant acid alpha-glucosidase polynucleotides or polypeptide sequences that do not include a signal peptide nucleotide or polypeptide sequence.

[0008] The present invention provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 2. In some embodiments, acid alpha-glucosidase is 27, 27 / 944, 28, 29 / 478, 30, 88, 107, 109, 109 / 842, 110, 113, 135, 137, 138, 148, 150, 247, 274, 276, 278, 375, 403, 414, 418, 418 / 499, 421, 426, 437, 444, 455, 463, 471 , includes at least one substitution in a position or set of positions selected from 471 / 478, 476, 489, 527, 547, 581, 610, 642, 668, 670, 692, 725 / 732, 750, 753, 786, 820, 862, 871, 895, 897, 930, 934, and 944, where these positions are numbered with reference to Sequence ID No. 2. In some embodiments, acid alpha-glucosidases include 27P, 27P / 944W, 27R, 28P, 28R, 28S, 29T / 478T, 30G, 30K, 30T, 88G, 88S, 107G, 107P, 109G / 842E, 109P, 110G, 110L, 113S, 135A, 135Q, 137P, 138A, 148G, 148Y, 150G, 247R, 274G, 276F, 276Y, 278A, 278G, 375E, 403W, 414P, 418 E / 499R, 418R, 421S, 426R, 437S, 444T, 455V, 463A, 471Q / 478S, 471S, 476A, 476H, 489R, 527R, 547G, 581G, 581T, 610A, 610G, 6 10S, 642M, 642Q, 642S, 668H, 670N, 692Q, 725N / 732I, 750P, 753T, 786P, 786Y, 820E, 862G, 871E, 895R, 897V, 930R, 934R, 944G,The acidic alpha-glucosidase includes at least one substitution or set of substitutions at one or more positions selected from and 944R, where these positions are numbered with reference to Sequence ID No. 2. In some embodiments, the acidic alpha-glucosidase includes F27P, F27P / C944W, F27R, L28P, L28R, L28S, L29T / A478T, V30G, V30K, V30T, K88G, K88S, Q107G, Q107P, L109G / G842E, L109P, Q110G, Q110L, Q1 13S, S135A, S135Q, E137P, M138A, T148G, T148Y, T150G, Q247R, D274G, A276F, A276Y, T 278A, T278G, I375E, R403W, R414P, A418E / H499R, A418R, Q421S, G426R, A437S, A444T, R It includes at least one substitution or set of substitutions at one or more positions selected from 455V, E463A, K471Q / A478S, K471S, S476A, S476H, A489R, N527R, A547G, K581G, K581T, W610A, W610G, W610S, L642M, L642Q, L642S, S668H, L670N, T692Q, K725N / V732I, A750P, A753T, R786P, R786Y, G820E, R862G, L871E, K895R, T897V, C930R, L934R, C944G, and C944R, where these positions are numbered with reference to Sequence ID No. 2. In some embodiments, acid alpha-glucosidase is 29 / 218 / 240 / 668 / 700 / 744 / 869, 29 / 218 / 240 / 700 / 869, 29 / 240 / 596 / 668 / 700 / 744 / 869, 29 / 240 / 596 / 668 / 869, 36 / 106 / 150 / 218 / 527 / 750 / 883 / 894, 106 / 112 / 150 / 218 / 414 / 527 / 793 / 883, 106 / 150 / 169 / 218 / 414 / 486 / 527 / 750 / 894, 106 / 150 / 169 / 218 / 414 / 486 / 527 / 894, 106 / 150 / 169 / 218 / 414 / 486 / 749 / 793 / 883 / 894, 106 / 150 / 169 / 218 / 414 / 486 / 750 / 793 / 883 / 894,106 / 150 / 169 / 218 / 414 / 486 / 793 / 883、106 / 150 / 169 / 218 / 414 / 486 / 894、106 / 150 / 169 / 218 / 414 / 749 / 750 / 793 / 883、106 / 150 / 169 / 218 / 414 / 749 / 793、106 / 150 / 169 / 218 / 414 / 749 / 793 / 883、106 / 150 / 169 / 218 / 486 / 527 / 749 / 793 / 894、106 / 150 / 169 / 218 / 486 / 749 / 883、106 / 150 / 169 / 218 / 486 / 883、106 / 150 / 169 / 218 / 749 / 800、106 / 150 / 169 / 414 / 486 / 749 / 750 / 883、106 / 150 / 169 / 527 / 749 / 793 / 883、106 / 150 / 169 / 749 / 793 / 883 / 894、106 / 150 / 218 / 331 / 414 / 486 / 527 / 733 / 749 / 793、106 / 150 / 218 / 414 / 486 / 642 / 750 / 793 / 883、106 / 150 / 218 / 414 / 486 / 750 / 793 / 894、106 / 150 / 218 / 414 / 527 / 749 / 750 / 883、106 / 150 / 218 / 414 / 527 / 749 / 793 / 883 / 894、106 / 150 / 218 / 414 / 749 / 750 / 793 / 883 / 894、106 / 150 / 218 / 414 / 749 / 793 / 883、106 / 150 / 218 / 486 / 527 / 749 / 894、106 / 150 / 218 / 486 / 793 / 883、106 / 150 / 218 / 527 / 749 / 750 / 793、106 / 150 / 218 / 527 / 793 / 894、106 / 150 / 218 / 749 / 750 / 793、106 / 150 / 218 / 793、106 / 150 / 218 / 793 / 894、106 / 150 / 245 / 793 / 883 / 894、106 / 150 / 414 / 749 / 750 / 793 / 894、106 / 150 / 414 / 749 / 793 / 894、106 / 150 / 486 / 527 / 750 / 793、106 / 150 / 486 / 749 / 793 / 883 / 894、106 / 150 / 749 / 793 / 883、106 / 169 / 185 / 218 / 414 / 749 / 750 / 793、106 / 191 / 280 / 402 / 414 / 444 / 727、106 / 191 / 414 / 444 / 522 / 928 / 944、106 / 191 / 414 / 489 / 928 / 944、106 / 280 / 402 / 414 / 444 / 489 / 727 / 944、150 / 169 / 218 / 414 / 527 / 793、150 / 218 / 414 / 486 / 749 / 750、150 / 218 / 414 / 486 / 750 / 793、150 / 218 / 414 / 486 / 750 / 793 / 883、150 / 218 / 414 / 749 / 750 / 793 / 894、150 / 218 / 414 / 749 / 793、150 / 218 / 527 / 749 / 793、150 / 218 / 749 / 750 / 793、150 / 218 / 749 / 793、150 / 414 / 486 / 527 / 750 / 894、150 / 414 / 486 / 749 / 750 / 793、150 / 486 / 750 / 883 / 894、169 / 486 / 750 / 793 / 883、180 / 275 / 402 / 518 / 547 / 610 / 638 / 669 / 671、180 / 402 / 431 / 507 / 547 / 610 / 669 / 671 / 793、180 / 402 / 507 / 547 / 610 / 671、191 / 280 / 402 / 414 / 444 / 465 / 842 / 928、191 / 280 / 402 / 414 / 444 / 489 / 500 / 944、191 / 280 / 414 / 444 / 489 / 500 / 522 / 842 / 928 / 944、191 / 280 / 414 / 444 / 489 / 522 / 727 / 944、191 / 280 / 414 / 489 / 842 / 928 / 944、191 / 280 / 414 / 944、191 / 414 / 522 / 842 / 944、196 / 402 / 431 / 547 / 610 / 638、218 / 668 / 700 / 869、224 / 402 / 507 / 518 / 547 / 638 / 668、269 / 275 / 431 / 518 / 547 / 638 / 668 / 669、275 / 281 / 402 / 431 / 507 / 518 / 610 / 668、275 / 281 / 402 / 431 / 518 / 547 / 610 / 669 / 671、275 / 281 / 402 / 507 / 518 / 547 / 638 / 669 / 671、275 / 281 / 402 / 518 / 547 / 610 / 638 / 671、275 / 281 / 402 / 518 / 547 / 610 / 668 / 669 / 887、275 / 281 / 402 / 547 / 610 / 638 / 669 / 671、275 / 281 / 431 / 518 / 547 / 638 / 669 / 671、275 / 281 / 507 / 547 / 669 / 671, 275 / 281 / 610 / 638 / 668 / 669, 275 / 281 / 671, 275 / 377 / 402 / 507 / 518 / 669 / 671 / 715, 275 / 402 / 431 / 507 / 547 / 671, 275 / 402 / 431 / 518 / 610 / 638 / 669 / 671 / 922, 275 / 402 / 507 / 547 / 610 / 638 / 668 / 669, 275 / 402 / 507 / 547 / 610 / 638 / 669 / 671, 275 / 402 / 507 / 547 / 610 / 671, 275 / 40 2 / 547 / 610 / 638 / 669 / 671, 275 / 402 / 547 / 638 / 669 / 671, 275 / 402 / 638 / 669 / 671, 275 / 431 / 507 / 518 / 547 / 668 / 669 / 671, 275 / 431 / 507 / 518 / 610 / 669 / 6 71, 275 / 431 / 507 / 547 / 610 / 638 / 671, 275 / 431 / 518 / 547 / 638 / 668, 275 / 431 / 518 / 610 / 638 / 669 / 671, 275 / 431 / 638, 275 / 507 / 518 / 547 / 610 / 638 / 668 / 6 69, 275 / 507 / 518 / 547 / 638 / 669 / 671, 275 / 507 / 547 / 610 / 638 / 669 / 671, 275 / 507 / 547 / 668 / 669 / 671, 275 / 518 / 671, 280 / 402 / 536 / 928, 281 / 402 / 507 / 518 / 547 / 610 / 638 / 669 / 671, 281 / 402 / 507 / 547 / 638 / 669 / 671, 281 / 402 / 518 / 547 / 610 / 638 / 668 / 669, 281 / 402 / 518 / 547 / 668, 281 / 431 / 507 / 518 / 547 / The position or set of positions selected from 610 / 638 / 668, 402 / 431 / 518 / 547 / 610 / 668, 402 / 431 / 518 / 547 / 671, 402 / 431 / 518 / 610, 402 / 431 / 547 / 638 / 671, 431 / 507 / 518 / 541 / 547 / 638 / 669 / 671, 431 / 507 / 518 / 669 / 671, 507 / 547 / 610, 507 / 547 / 638 / 669 / 671, 547 / 610 / 638 / 671, and 547 / 638 / 668 includes at least one substitution, where these positions areNumbered with reference to Sequence ID No. 2. In some embodiments, acid alpha-glucosidase is 29Q / 218S / 240I / 668D / 700F / 744V / 869L, 29Q / 240I / 596P / 668D / 869L, 29Q / 240I / 596S / 668D / 700F / 744V / 869T, 29V / 218S / 240I / 700F / 869T, 36R / 106P / 150S / 218S / 527D / 750P / 883H / 894R, 106P / 112S / 150S / 218S / 414G / 52, 7D / 793K / 883H、106P / 150S / 169S / 218S / 414G / 486E / 527D / 750P / 894R、106P / 150S / 169S / 218S / 414G / 486E / 527D / 894R、106P / 150S / 169S / 218S / 414G / 486E / 749E / 793K / 883H / 894R、106P / 150S / 169S / 218S / 414G / 486E / 750P / 793K / 883H / 894R、106P / 150S / 169S / 218S / 414G / 486E / 793K / 883H、106P / 150S / 169S / 218S / 414G / 486E / 894R、106P / 150S / 169S / 218S / 414G / 749E / 750P / 793K / 883H、106P / 150S / 169S / 218S / 414G / 749E / 793K、106P / 150S / 169S / 218S / 414G / 749E / 793K / 883H、106P / 150S / 169S / 218S / 486E / 527D / 749E / 793K / 894R、106P / 150S / 169S / 218S / 486E / 749E / 883H、106P / 150S / 169S / 218S / 486E / 883H、106P / 150S / 169S / 218S / 749E / 800A、106P / 150S / 169S / 414G / 486E / 749E / 750P / 883H、106P / 150S / 169S / 527D / 749E / 793K / 883H、106P / 150S / 169S / 749E / 793K / 883H / 894R、106P / 150S / 218S / 331A / 414G / 486E / 527D / 733E / 749E / 793K、106P / 150S / 218S / 414G / 486E / 642F / 750P / 793K / 883H、106P / 150S / 218S / 414G / 486E / 750P / 793K / 894R、106P / 150S / 218S / 414G / 527D / 749E / 750P / 883H、106P / 150S / 218S / 414G / 527D / 749E / 793K / 883H / 894G、106P / 150S / 218S / 414G / 749E / 750P / 793K / 883H / 894R、106P / 150S / 218S / 414G / 749E / 793K / 883H、106P / 150S / 218S / 486E / 527D / 749E / 894R、106P / 150S / 218S / 486E / 793K / 883H、106P / 150S / 218S / 527D / 749E / 750P / 793K、106P / 150S / 218S / 527D / 793K / 894G、106P / 150S / 218S / 749E / 750P / 793K、106P / 150S / 218S / 793K、106P / 150S / 218S / 793K / 894R、106P / 150S / 245S / 793K / 883H / 894R、106P / 150S / 414G / 749E / 750P / 793K / 894R、106P / 150S / 414G / 749E / 793K / 894R、106P / 150S / 486E / 527D / 750P / 793K、106P / 150S / 486E / 749E / 793K / 883H / 894G、106P / 150S / 749E / 793K / 883H、106P / 169S / 185G / 218S / 414G / 749E / 750P / 793K、106P / 191R / 280D / 402A / 414G / 444P / 727P、106P / 191R / 414G / 444P / 522V / 928T / 944S、106P / 191R / 414G / 489D / 928T / 944S、106P / 280D / 402A / 414G / 444P / 489D / 727P / 944S、150S / 169S / 218S / 414G / 527D / 793K、150S / 218S / 414G / 486A / 750P / 793K、150S / 218S / 414G / 486E / 749E / 750P、150S / 218S / 414G / 486E / 750P / 793K / 883H、150S / 218S / 414G / 749E / 750P / 793K / 894R、150S / 218S / 414G / 749E / 793K、150S / 218S / 527D / 749E / 793K、150S / 218S / 749E / 750P / 793K、150S / 218S / 749E / 793K、150S / 414G / 486E / 527D / 750P / 894R、150S / 414G / 486E / 749E / 750P / 793K、150S / 486E / 750P / 883H / 894G、169S / 486E / 750P / 793K / 883H、180H / 275M / 402A / 518V / 547G / 610R / 638I / 669H / 671N、180H / 402A / 431V / 507L / 547G / 610R / 669H / 671N / 793G、180H / 402A / 507L / 547G / 610R / 671N、191R / 280D / 402A / 414G / 444P / 465E / 842S / 928T、191R / 280D / 402A / 414G / 444P / 489D / 500A / 944S、191R / 280D / 414G / 444P / 489D / 500A / 522V / 842S / 928T / 944S、191R / 280D / 414G / 444P / 489D / 522V / 727P / 944S、191R / 280D / 414G / 489D / 842S / 928T / 944S、191R / 280D / 414G / 944S、191R / 414G / 522V / 842S / 944S、196V / 402A / 431V / 547G / 610R / 638I、218S / 668D / 700F / 869T、224F / 402A / 507L / 518V / 547G / 638I / 668D、269N / 275M / 431V / 518V / 547G / 638I / 668D / 669H、275M / 281V / 402A / 431V / 507L / 518V / 610R / 668D、275M / 281V / 402A / 507L / 518V / 547G / 638I / 669H / 671N、275M / 281V / 402A / 518V / 547G / 610R / 638I / 671N、275M / 281V / 402A / 518V / 547G / 610R / 668D / 669H / 887D、275M / 281V / 402A / 547G / 610R / 638I / 669H / 671N、275M / 281V / 507L / 547G / 669H / 671N、275M / 281V / 610R / 638I / 668D / 669H、275M / 402A / 431V / 507L / 547G / 671N、275M / 402A / 507L / 547G / 610R / 671N、275M / 402A / 547G / 638I / 669H / 671N、275M / 431V / 518V / 547G / 638I / 668D、275M / 431V / 518V / 610R / 638I / 669H / 671N、275M / 431V / 638I、275M / 507L / 547G / 668D / 669H / 671N、275V / 281V / 402A / 431V / 518V / 547G / 610R / 669H / 671N、275V / 281V / 431V / 518V / 547G / 638I / 669H / 671N、275V / 281V / 671N、275V / 377K / 402A / 507L / 518V / 669H / 671N / 715G, 275V / 402A / 431V / 518V / 610R / 638I / 669H / 671N / 922L, 275V / 402A / 507L / 547G / 610R / 638I / 668D / 6 69H, 275V / 402A / 507L / 547G / 610R / 638I / 669H / 671N, 275V / 402A / 547G / 61 0R / 638I / 669H / 671N, 275V / 402A / 638I / 669H / 671N, 275V / 431V / 507L / 518 V / 547G / 668D / 669H / 671N, 275V / 431V / 507L / 518V / 610R / 669H / 671N, 275V / 431V / 507L / 547G / 610R / 638I / 671N, 275V / 507L / 518V / 547G / 610R / 638I / 668D / 669H, 275V / 507L / 518V / 547G / 638I / 669H / 671N, 275V / 507L / 547G / 6 10R / 638I / 669H / 671N, 275V / 518V / 671N, 280D / 402A / 536I / 928T, 281V / 40 2A / 507L / 518V / 547G / 610R / 638I / 669H / 671N, 281V / 402A / 507L / 547G / 638 I / 669H / 671N, 281V / 402A / 518V / 547G / 610R / 638I / 668D / 669H, 281V / 402A / 518V / 547G / 668D, 281V / 431V / 507L / 518V / 547G / 610R / 638I / 668D, 402A / 431V / 518V / 547G / 610R / 668D, 402A / 431V / 518V / 547G / 671N, 402A / 431V / 5 The alpha-glucosidase includes at least one substitution or set of substitutions at one or more positions selected from 18V / 610R, 402A / 431V / 547G / 638I / 671N, 431V / 507L / 518V / 541E / 547G / 638I / 669H / 671N, 431V / 507L / 518V / 669H / 671N, 507L / 547G / 610R, 507L / 547G / 638I / 669H / 671N, 547G / 610R / 638I / 671N, and 547G / 638I / 668D, where these positions are numbered with reference to Sequence ID No. 2. In some embodiments, the acid alpha-glucosidase isL29Q / L218S / L240I / S668D / H700F / I744V / I869L、L29Q / L240I / A596P / S668D / I869L、L29Q / L240I / A596S / S668D / H700F / I744V / I869T、L29V / L218S / L240I / H700F / I869T、G36R / K106P / T150S / L218S / N527D / A750P / R883H / Q894R、K106P / A112S / T150S / L218S / R414G / N527D / E793K / R883H、K106P / T150S / N169S / L218S / R414G / T486E / N527D / A750P / Q894R、K106P / T150S / N169S / L218S / R414G / T486E / N527D / Q894R、K106P / T150S / N169S / L218S / R414G / T486E / Q749E / E793K / R883H / Q894R、K106P / T150S / N169S / L218S / R414G / T486E / A750P / E793K / R883H / Q894R、K106P / T150S / N169S / L218S / R414G / T486E / E793K / R883H、K106P / T150S / N169S / L218S / R414G / T486E / Q894R、K106P / T150S / N169S / L218S / R414G / Q749E / A750P / E793K / R883H、K106P / T150S / N169S / L218S / R414G / Q749E / E793K、K106P / T150S / N169S / L218S / R414G / Q749E / E793K / R883H、K106P / T150S / N169S / L218S / T486E / N527D / Q749E / E793K / Q894R、K106P / T150S / N169S / L218S / T486E / Q749E / R883H、K106P / T150S / N169S / L218S / T486E / R883H、K106P / T150S / N169S / L218S / Q749E / P800A、K106P / T150S / N169S / R414G / T486E / Q749E / A750P / R883H、K106P / T150S / N169S / N527D / Q749E / E793K / R883H、K106P / T150S / N169S / Q749E / E793K / R883H / Q894R、K106P / T150S / L218S / V331A / R414G / T486E / N527D / D733E / Q749E / E793K、K106P / T150S / L218S / R414G / T486E / L642F / A750P / E793K / R883H、K106P / T150S / L218S / R414G / T486E / A750P / E793K / Q894R、K106P / T150S / L218S / R414G / 、 N527D / Q749E / A750P / R883H、K106P / T150S / L218S / R414G / N527D / Q749E / E793K / R883H / Q894G、K106P / T150S / L218S / R414G / Q749E / A750P / E793K / R883H / Q894R、K106P / T150S / L218S / R414G / Q749E / E793K / R883H、K106P / T150S / L218S / T486E / N527D / Q749E / Q894R、K106P / T150S / L218S / T486E / E793K / R883H、K106P / T150S / L218S / N527D / Q749E / A750P / E793K、K106P / T150S / L218S / N527D / E793K / Q894G、K106P / T150S / L218S / Q749E / A750P / E793K、K106P / T150S / L218S / E793K、K106P / T150S / L218S / E793K / Q894R、K106P / T150S / P245S / E793K / R883H / Q894R、K106P / T150S / R414G / Q749E / A750P / E793K / Q894R、K106P / T150S / R414G / Q749E / E793K / Q894R、K106P / T150S / T486E / N527D / A750P / E793K、K106P / T150S / T486E / Q749E / E793K / R883H / Q894G、K106P / T150S / Q749E / E793K / R883H、K106P / N169S / V185G / L218S / R414G / Q749E / A750P / E793K、K106P / H191R / G280D / S402A / R414G / A444P / S727P、K106P / H191R / R414G / A444P / E522V / D928T / C944S、K106P / H191R / R414G / A489D / D928T / C944S、K106P / G280D / S402A / R414G / A444P / A489D / S727P / C944S、T150S / N169S / L218S / R414G / N527D / E793K、T150S / L218S / R414G / T486A / A750P / E793K、T150S / L218S / R414G / T486E / Q749E / A750P、T150S / L218S / R414G / T486E / A750P / E793K / R883H、T150S / L218S / R414G / Q749E / A750P / E793K / Q894R、T150S / L218S / R414G / Q749E / E793K、T150S / L218S / N527D / Q749E / E793K、T150S / L218S / Q749E / A750P / E793K、T150S / L218S / Q749E / E793K、T150S / R414G / T486E / N527D / A750P / Q894R、T150S / R414G / T486E / Q749E / A750P / E793K、T150S / T486E / A750P / R883H / Q894G、N169S / T486E / A750P / E793K / R883H、N180H / L275M / S402A / I518V / A547G / W610R / V638I / L669H / S671N、N180H / S402A / M431V / M507L / A547G / W610R / L669H / S671N / E793G、N180H / S402A / M507L / A547G / W610R / S671N、H191R / G280D / S402A / R414G / A444P / G465E / G842S / D928T、H191R / G280D / S402A / R414G / A444P / A489D / D500A / C944S、H191R / G280D / R414G / A444P / A489D / D500A / E522V / G842S / D928T / C944S、H191R / G280D / R414G / A444P / A489D / E522V / S727P / C944S、H191R / G280D / R414G / A489D / G842S / D928T / C944S、H191R / G280D / R414G / C944S、H191R / R414G / E522V / G842S / C944S、A196V / S402A / M431V / A547G / W610R / V638I、L218S / S668D / H700F / I869T、L224F / S402A / M507L / I518V / A547G / V638I / S668D、T269N / L275M / M431V / I518V / A547G / V638I / S668D / L669H、L275M / A281V / S402A / M431V / M507L / I518V / W610R / S668D、L275M / A281V / S402A / M507L / I518V / A547G / V638I / L669H / S671N、L275M / A281V / S402A / I518V / A547G / W610R / V638I / S671N、L275M / A281V / S402A / I518V / A547G / W610R / S668D / L669H / E887D、L275M / A281V / S402A / A547G / W610R / V638I / L669H / S671N、L275M / A281V / M507L / A547G / L669H / S671N、L275M / A281V / W610R / V638I / S668D / L669H、L275M / S402A / M431V / M507L / A547G / S671N、L275M / S402A / M507L / A547G / W610R / S671N、L275M / S402A / A547G / V638I / L669H / S671N、L275M / M431V / I518V / A547G / V638I / S668D、L275M / M431V / I518V / W610R / V638I / L669H / S671N、L275M / M431V / V638I、L275M / M507L / A547G / S668D / L669H / S671N、L275V / A281V / S402A / M431V / I518V / A547G / W610R / L669H / S671N、L275V / A281V / M431V / I518V / A547G / V638I / L669H / S671N、L275V / A281V / S671N、L275V / R377K / S402A / M507L / I518V / L669H / S671N / V715G、L275V / S402A / M431V / I518V / W610R / V638I / L669H / S671N / P922L、L275V / S402A / M507L / A547G / W610R / V638I / S668D / L669H、L275V / S402A / M507L / A547G / W610R / V638I / L669H / S671N、L275V / S402A / A547G / W610R / V638I / L669H / S671N、L275V / S402A / V638I / L669H / S671N、L275V / M431V / M507L / I518V / A547G / S668D / L669H / S671N、L275V / M431V / M507L / I518V / W610R / L669H / S671N、L275V / M431V / M507L / A547G / W610R / V638I / S671N, L275V / M507L / I518V / A547G / W610R / V638I / S668D / L669H, L275V / M50 7L / I518V / A547G / V638I / L669H / S671N, L275V / M507L / A547G / W610R / V638I / L669H / S671N, L275V / I518V / S671N, G280D / S 402A / V536I / D928T, A281V / S402A / M507L / I518V / A547G / W610R / V638I / L669H / S671N, A281V / S402A / M507L / A547G / V638 I / L669H / S671N, A281V / S402A / I518V / A547G / W610R / V638I / S668D / L669H, A281V / S402A / I518V / A547G / S668D, A281V / M4 31V / M507L / I518V / A547G / W610R / V638I / S668D, S402A / M431V / I518V / A547G / W610R / S668D, S402A / M431V / I518V / A547G / S671N, S402A / M431V / I518V / W610R, S402A / M431V / A547G / V638I / S671N, M431V / M507L / I518V / G541E / A547G / V638I / L66 The following positions are included: 9H / S671N, M431V / M507L / I518V / L669H / S671N, M507L / A547G / W610R, M507L / A547G / V638I / L669H / S671N, A547G / W610R / V638I / S671N, and A547G / V638I / S668D, each of which is numbered with reference to Sequence ID No. 2. In some embodiments, acid alpha-glucosidase is 27, 27 / 944, 28, 29 / 218 / 240 / 668 / 700 / 744 / 869, 29 / 218 / 240 / 700 / 869, 29 / 240 / 596 / 668 / 700 / 744 / 869, 29 / 240 / 596 / 668 / 869, 29 / 478, 30, 36 / 106 / 150 / 218 / 527 / 750 / 883 / 894, 106 / 112 / 150 / 218 / 414 / 527 / 793 / 883,106 / 150 / 169 / 218 / 414 / 486 / 527 / 750 / 894、106 / 150 / 169 / 218 / 414 / 486 / 527 / 894、106 / 150 / 169 / 218 / 414 / 486 / 749 / 793 / 883 / 894、106 / 150 / 169 / 218 / 414 / 486 / 750 / 793 / 883 / 894、106 / 150 / 169 / 218 / 414 / 486 / 793 / 883、106 / 150 / 169 / 218 / 414 / 486 / 894、106 / 150 / 169 / 218 / 414 / 749 / 750 / 793 / 883、106 / 150 / 169 / 218 / 414 / 749 / 793、106 / 150 / 169 / 218 / 414 / 749 / 793 / 883、106 / 150 / 169 / 218 / 486 / 749 / 883、106 / 150 / 169 / 218 / 486 / 883、106 / 150 / 169 / 414 / 486 / 749 / 750 / 883、106 / 150 / 169 / 527 / 749 / 793 / 883、106 / 150 / 169 / 749 / 793 / 883 / 894、106 / 150 / 218 / 331 / 414 / 486 / 527 / 733 / 749 / 793、106 / 150 / 218 / 414 / 486 / 642 / 750 / 793 / 883、106 / 150 / 218 / 414 / 486 / 750 / 793 / 894、106 / 150 / 218 / 414 / 527 / 749 / 750 / 883、106 / 150 / 218 / 414 / 527 / 749 / 793 / 883 / 894、106 / 150 / 218 / 414 / 749 / 750 / 793 / 883 / 894、106 / 150 / 218 / 414 / 749 / 793 / 883、106 / 150 / 218 / 486 / 527 / 749 / 894、106 / 150 / 218 / 486 / 793 / 883、106 / 150 / 218 / 527 / 793 / 894、106 / 150 / 245 / 793 / 883 / 894、106 / 150 / 414 / 749 / 750 / 793 / 894、106 / 150 / 414 / 749 / 793 / 894、106 / 150 / 486 / 527 / 750 / 793、106 / 150 / 486 / 749 / 793 / 883 / 894、106 / 150 / 749 / 793 / 883、106 / 169 / 185 / 218 / 414 / 749 / 750 / 793、106 / 191 / 280 / 402 / 414 / 444 / 727、106 / 280 / 402 / 414 / 444 / 489 / 727 / 944、107、109、109 / 842、110、135、138、148、150、150 / 218 / 414 / 486 / 749 / 750、150 / 218 / 414 / 486 / 750 / 793、150 / 218 / 414 / 486 / 750 / 793 / 883、150 / 218 / 414 / 749 / 750 / 793 / 894、150 / 218 / 414 / 749 / 793、150 / 414 / 486 / 527 / 750 / 894、150、 / 414 / 486 / 749 / 750 / 793、150 / 486 / 750 / 883 / 894、169 / 486 / 750 / 793 / 883、180 / 275 / 402 / 518 / 547 / 610 / 638 / 669 / 671、180 / 402 / 431 / 507 / 547 / 610 / 669 / 671 / 793、180 / 402 / 507 / 547 / 610 / 671、191 / 280 / 402 / 414 / 444 / 489 / 500 / 944、191 / 280 / 414 / 444 / 489 / 522 / 727 / 944、191 / 280 / 414 / 944、191 / 414 / 522 / 842 / 944、196 / 402 / 431 / 547 / 610 / 638、218 / 668 / 700 / 869、224 / 402 / 507 / 518 / 547 / 638 / 668、269 / 275 / 431 / 518 / 547 / 638 / 668 / 669、274、275 / 281 / 402 / 431 / 507 / 518 / 610 / 668、275 / 281 / 402 / 431 / 518 / 547 / 610 / 669 / 671、275 / 281 / 402 / 507 / 518 / 547 / 638 / 669 / 671、275 / 281 / 402 / 518 / 547 / 610 / 638 / 671、275 / 281 / 402 / 518 / 547 / 610 / 668 / 669 / 887、275 / 281 / 402 / 547 / 610 / 638 / 669 / 671、275 / 281 / 431 / 518 / 547 / 638 / 669 / 671、275 / 281 / 507 / 547 / 669 / 671、275 / 281 / 610 / 638 / 668 / 669、275 / 377 / 402 / 507 / 518 / 669 / 671 / 715、275 / 402 / 431 / 507 / 547 / 671、275 / 402 / 431 / 518 / 610 / 638 / 669 / 671 / 922、275 / 402 / 507 / 547 / 610 / 638 / 668 / 669、275 / 402 / 507 / 547 / 610 / 638 / 669 / 671、275 / 402 / 507 / 547 / 610 / 671、275 / 402 / 547 / 610 / 638 / 669 / 671、275 / 402 / 547 / 638 / 669 / 671、275 / 402 / 638 / 669 / 671、275 / 431 / 507 / 518 / 547 / 668 / 669 / 671、275 / 431 / 507 / 518 / 610 / 669 / 671、275 / 431 / 507 / 547 / 610 / 638 / 671、275 / 431 / 518 / 547 / 638 / 668, 275 / 431 / 518 / 610 / 638 / 669 / 671, 275 / 431 / 638, 275 / 507 / 518 / 547 / 610 / 638 / 668 / 669, 275 / 507 / 518 / 547 / 638 / 669 / 671, 275 / 507 / 547 / 610 / 638 / 669 / 671, 275 / 507 / 547 / 6 68 / 669 / 671, 276, 281 / 402 / 507 / 518 / 547 / 610 / 638 / 669 / 671, 281 / 402 / 507 / 547 / 638 / 669 / 671, 281 / 402 / 518 / 547 / 610 / 638 / 668 / 669, 281 / 402 / 518 / 547 / 668, 281 / 431 / 507 / 518 / 547 / 610 / 638 / 668, 375 ,402 / 431 / 518 / 547 / 610 / 668,402 / 431 / 518 / 547 / 671,402 / 431 / 518 / 610,402 / 431 / 547 / 638 / 671,403,414,418 / 499,431 / 507 / 518 / 541 / 547 / 638 / 669 / 671,431 / 507 / 518 / 669 / 671,437,471 / 478,507 / It includes at least one substitution or set of substitutions at one or more positions selected from 547 / 610, 507 / 547 / 638 / 669 / 671, 547, 547 / 610 / 638 / 671, 547 / 638 / 668, 581, 642, 670, 692, 750, 753, 820, 871, and 944, where these positions are numbered with reference to Sequence ID No. 2. In some embodiments, acid alpha-glucosidase is 27P, 27P / 944W, 27R, 28P, 28S, 29Q / 218S / 240I / 668D / 700F / 744V / 869L, 29Q / 240I / 596P / 668D / 869L, 29Q / 240I / 596S / 668D / 700F / 744V / 869T, 29T / 478T, 29V / 218S / 240I / 700F / 869T, 30G, 30K, 30T, 36R / 106P / 150S / 218S / 527D / 750P / 883H / 894R, 106P / 11 2S / 150S / 218S / 414G / 527D / 793K / 883H, 106P / 150S / 169S / 218S / 414G / 486E / 527D / 750P / 894R,106P / 150S / 169S / 218S / 414G / 486E / 527D / 894R、106P / 150S / 169S / 218S / 414G / 486E / 749E / 793K / 883H / 894R、106P / 150S / 169S / 218S / 414G / 486E / 750P / 793K / 883H / 894R、106P / 150S / 169S / 218S / 414G / 486E / 793K / 883H、106P / 150S / 169S / 218S / 414G / 486E / 894R、106P / 150S / 169S / 218S / 414G / 749E / 750P / 793K / 883H、106P / 150S / 169S / 218S / 414G / 749E / 793K、106P / 150S / 169S / 218S / 414G / 749E / 793K / 883H、106P / 150S / 169S / 218S / 486E / 749E / 883H、106P / 150S / 169S / 218S / 486E / 883H、106P / 150S / 169S / 414G / 486E / 749E / 750P / 883H、106P / 150S / 169S / 527D / 749E / 793K / 883H、106P / 150S / 169S / 749E / 793K / 883H / 894R、106P / 150S / 218S / 331A / 414G / 486E / 527D / 733E / 749E / 793K、106P / 150S / 218S / 414G / 486E / 642F / 750P / 793K / 883H、106P / 150S / 218S / 414G / 486E / 750P / 793K / 894R、106P / 150S / 218S / 414G / 527D / 749E / 750P / 883H、106P / 150S / 218S / 414G / 527D / 749E / 793K / 883H / 894G、106P / 150S / 218S / 414G / 749E / 750P / 793K / 883H / 894R、106P / 150S / 218S / 414G / 749E / 793K / 883H、106P / 150S / 218S / 486E / 527D / 749E / 894R、106P / 150S / 218S / 486E / 793K / 883H、106P / 150S / 218S / 527D / 793K / 894G、106P / 150S / 245S / 793K / 883H / 894R、106P / 150S / 414G / 749E / 750P / 793K / 894R、106P / 150S / 414G / 749E / 793K / 894R、106P / 150S / 486E / 527D / 750P / 793K、106P / 150S / 486E / 749E / 793K / 883H / 894G、106P / 150S / 749E / 793K / 883H、106P / 169S / 185G / 218S / 414G / 749E / 750P / 793K、106P / 191R / 280D / 402A / 414G / 444P / 727P、106P / 280D / 402A / 414G / 444P / 489D / 727P / 944S、107G、109G / 842E、109P、110G、110L、135Q、138A、148G、148Y、150G、150S / 218S / 414G / 486A / 750P / 793K、150S / 218S / 414G / 486E / 749E / 750P、150S / 218S / 414G / 486E / 750P / 793K / 883H、150S / 218S / 414G / 749E / 750P / 793K / 894R、150S / 218S / 414G / 749E / 793K、150S / 414G / 486E / 527D / 750P / 894R、150S / 414G / 486E / 749E / 750P / 793K、150S / 486E / 750P / 883H / 894G、169S / 486E / 750P / 793K / 883H、180H / 275M / 402A / 518V / 547G / 610R / 638I / 669H / 671N、180H / 402A / 431V / 507L / 547G / 610R / 669H / 671N / 793G、180H / 402A / 507L / 547G / 610R / 671N、191R / 280D / 402A / 414G / 444P / 489D / 500A / 944S、191R / 280D / 414G / 444P / 489D / 522V / 727P / 944S、191R / 280D / 414G / 944S、191R / 414G / 522V / 842S / 944S、196V / 402A / 431V / 547G / 610R / 638I、218S / 668D / 700F / 869T、224F / 402A / 507L / 518V / 547G / 638I / 668D、269N / 275M / 431V / 518V / 547G / 638I / 668D / 669H、274G、275M / 281V / 402A / 431V / 507L / 518V / 610R / 668D、275M / 281V / 402A / 507L / 518V / 547G / 638I / 669H / 671N、275M / 281V / 402A / 518V / 547G / 610R / 638I / 671N、275M / 281V / 402A / 518V / 547G / 610R / 668D / 669H / 887D、275M / 281V / 402A / 547G / 610R / 638I / 669H / 671N、275M / 281V / 507L / 547G / 669H / 671N、275M / 281V / 610R / 638I / 668D / 669H、275M / 402A / 431V / 507L / 547G / 671N、275M / 402A / 507L / 547G / 610R / 671N、275M / 402A / 547G / 638I / 669H / 671N、275M / 431V / 518V / 547G / 638I / 668D、275M / 431V / 518V / 610R / 638I / 669H / 671N、275M / 431V / 638I、275M / 507L / 547G / 668D / 669H / 671N、275V / 281V / 402A / 431V / 518V / 547G / 610R / 669H / 671N、275V / 281V / 431V / 518V / 547G / 638I / 669H / 671N、275V / 377K / 402A / 507L / 518V / 669H / 671N / 715G、275V / 402A / 431V / 518V / 610R / 638I / 669H / 671N / 922L、275V / 402A / 507L / 547G / 610R / 638I / 668D / 669H、275V / 402A / 507L / 547G / 610R / 638I / 669H / 671N、275V / 402A / 547G / 610R / 638I / 669H / 671N、275V / 402A / 638I / 669H / 671N、275V / 431V / 507L / 518V / 547G / 668D / 669H / 671N、275V / 431V / 507L / 518V / 610R / 669H / 671N、275V / 431V / 507L / 547G / 610R / 638I / 671N、275V / 507L / 518V / 547G / 610R / 638I / 668D / 669H、275V / 507L / 518V / 547G / 638I / 669H / 671N、275V / 507L / 547G / 610R / 638I / 669H / 671N、276Y、281V / 402A / 507L / 518V / 547G / 610R / 638I / 669H / 671N、281V / 402A / 507L / 547G / 638I / 669H / 671N、281V / 402A / 518V / 547G / 610R / 638I / 668D / 669H、281V / 402A / 518V / 547G / 668D、281V / 431V / 507L / 518V / 547G / 610R / 638I / 668D、375E、402A / 431V / 518V / 547G / 610R / 668D、402A / 431V / 518V / 547G / 671N、402A / 431V / 51、 8V / 610R, 402A / 431V / 547G / 638I / 671N, 403W, 414P, 418E / 499R, 431V / 507L / 518V / 541E / 547G / 638I / 6 69H / 671N, 431V / 507L / 518V / 669H / 671N, 437S, 471Q / 478S, 507L / 547G / 610R, 507L / 547G / 638I / 669H / 6 It includes at least one substitution or set of substitutions at one or more positions selected from 71N, 547G, 547G / 610R / 638I / 671N, 547G / 638I / 668D, 581G, 581T, 642Q, 642S, 670N, 692Q, 750P, 753T, 820E, 871E, and 944G, where these positions are numbered with reference to Sequence ID No. 2. In some embodiments, the acid alpha-glucosidase is F27P, F27P / C944W, F27R, L28P, L28S, L29Q / L218S / L240I / S668D / H700F / I744V / I869L, L29Q / L240I / A596P / S668D / I869L, L29Q / L240I / A596S / S668D / H700F / I 744V / I869T, L29T / A478T, L29V / L218S / L240I / H700F / I869T, V30G, V30K, V30T, G36R / K106P / T1 50S / L218S / N527D / A750P / R883H / Q894R, K106P / A112S / T150S / L218S / R414G / N527D / E793K / R88 3H, K106P / T150S / N169S / L218S / R414G / T486E / N527D / A750P / Q894R, K106P / T150S / N169S / L21 8S / R414G / T486E / N527D / Q894R, K106P / T150S / N169S / L218S / R414G / T486E / Q749E / E793K / R883 H / Q894R, K106P / T150S / N169S / L218S / R414G / T486E / A750P / E793K / R883H / Q894R, K106P / T150S / N169S / L218S / R414G / T486E / E793K / R883H, K106P / T150S / N169S / L218S / R414G / T486E / Q894R,K106P / T150S / N169S / L218S / R414G / Q749E / A750P / E793K / R883H、K106P / T150S / N169S / L218S / R414G / Q749E / E793K、K106P / T150S / N169S / L218S / R414G / Q749E / E793K / R883H、K106P / T150S / N169S / L218S / T486E / Q749E / R883H、K106P / T150S / N169S / L218S / T486E / R883H、K106P / T150S / N169S / R414G / T486E / Q749E / A750P / R883H、K106P / T150S / N169S / N527D / Q749E / E793K / R883H、K106P / T150S / N169S / Q749E / E793K / R883H / Q894R、K106P / T150S / L218S / V331A / R414G / T486E / N527D / D733E / Q749E / E793K、K106P / T150S / L218S / R414G / T486E / L642F / A750P / E793K / R883H、K106P / T150S / L218S / R414G / T486E / A750P / E793K / Q894R、K106P / T150S / L218S / R414G / N527D / Q749E / A750P / R883H、K106P / T150S / L218S / R414G / N527D / Q749E / E793K / R883H / Q894G、K106P / T150S / L218S / R414G / Q749E / A750P / E793K / R883H / Q894R、K106P / T150S / L218S / R414G / Q749E / E793K / R883H、K106P / T150S / L218S / T486E / N527D / Q749E / Q894R、K106P / T150S / L218S / T486E / E793K / R883H、K106P / T150S / L218S / N527D / E793K / Q894G、K106P / T150S / P245S / E793K / R883H / Q894R、K106P / T150S / R414G / Q749E / A750P / E793K / Q894R、K106P / T150S / R414G / Q749E / E793K / Q894R、K106P / T150S / T486E / N527D / A750P / E793K、K106P / T150S / T486E / Q749E / E793K / R883H / Q894G、K106P / T150S / Q749E / E793K / R883H、K106P / N169S / V185G / L218S / R414G / Q749E / A750P / E793K、K106P / H191R / G280D / S402A / R414G / A444P / S727P、K106P / G280D / S402A / R414G / A444P / A489D / S727P / C944S、Q107G、L109G / G842E、L109P、Q110G、Q110L、S135Q、M138A、T148G、T148Y、T150G、T150S / L218S / R414G / T486A / A750P / E793K、T150S / L218S / R414G / T486E / Q749E / A750P、T150S / L218S / R414G / T486E / A750P / E793K / R883H、T150S / L218S / R414G / Q749E / A750P / E793K / Q894R、T150S / L218S / R414G / Q749E / E793K、T150S / R414G / T486E / N527D / A750P / Q894R、T150S / R414G / T486E / Q749E / A750P / E793K、T150S / T486E / A750P / R883H / Q894G、N169S / T486E / A750P / E793K / R883H、N180H / L275M / S402A / I518V / A547G / W610R / V638I / L669H / S671N、N180H / S402A / M431V / M507L / A547G / W610R / L669H / S671N / E793G、N180H / S402A / M507L / A547G / W610R / S671N、H191R / G280D / S402A / R414G / A444P / A489D / D500A / C944S、H191R / G280D / R414G / A444P / A489D / E522V / S727P / C944S、H191R / G280D / R414G / C944S、H191R / R414G / E522V / G842S / C944S、A196V / S402A / M431V / A547G / W610R / V638I、L218S / S668D / H700F / I869T、L224F / S402A / M507L / I518V / A547G / V638I / S668D、T269N / L275M / M431V / I518V / A547G / V638I / S668D / L669H、D274G、L275M / A281V / S402A / M431V / M507L / I518V / W610R / S668D、L275M / A281V / S402A / M50 7L / I518V / A547G / V638I / L669H / S671N、L275M / A281V / S402A / I518V / A547G / W610R / V638I / S671N、L275M / A281V / S402A / I518V / A547G / W610R / S668D / L 669H / E887D, L275M / A281V / S402A / A547G / W610R / V638I / L669H / S671N, L275M / A281V / M507L / A547G / L669H / S671N, L275M / A281V / W610R / V638I / S668D / L669H、L275M / S402A / M431V / M507L / A547G / S671N、L275M / S402A / M507L / A547G / W610R / S671N、L275M / S402A / A547G / V638I / L669H / S671N、L275M / M43 1V / I518V / A547G / V638I / S668D、L275M / M431V / I518V / W610R / V638I / L669 H / S671N、L275M / M431V / V638I、L275M / M507L / A547G / S668D / L669H / S671N、 L275V / A281V / S402A / M431V / I518V / A547G / W610R / L669H / S671N, L275V / A281V / M431V / I518V / A547G / V638I / L669H / S671N, L275V / R377K / S402A / M507 L / I518V / L669H / S671N / V715G, L275V / S402A / M431V / I518V / W610R / V638I / L669H / S671N / P922L, L275V / S402A / M507L / A547G / W610R / V638I / S668D / L6 69H, L275V / S402A / M507L / A547G / W610R / V638I / L669H / S671N, L275V / S402A / A547G / W610R / V638I / L669H / S671N, L275V / S402A / V638I / L669H / S671NOne or more substitutions or sets of substitutions at one or more positions selected from L275V / M431V / M507L / I518V / A547G / S668D / L669H / S671N, L275V / M431V / M507L / I518V / W610R / L669H / S671N, L275V / M431V / M507L / A547G / W610R / V638I / S671N, L275V / M507L / I518V / A547G / W610R / V638I / S668D / L669H, L275V / M507L / I518V / A547G / V638I / L669H / S671N, L275V / M507L / A547G / W610R / V638I / L669H / S671N, A276Y, A281V / S402A / M507L / I518V / A547G / W610R / V638I / L669H / S671N, A281V / S402A / M507L / A547G / V638I / L669H / S671N, A281V / S402A / I518V / A547G / W610R / V638I / S668D / L669H, A281V / S402A / I518V / A547G / S668D, A281V / M431V / M507L / I518V / A547G / W610R / V638I / S668D, I375E, S402A / M431V / I518V / A547G / W610R / S668D, S402A / M431V / I518V / A547G / S671N, S402A / M431V / I518V / W610R, S402A / M431V / A547G / V638I / S671N, R403W, R414P, A418E / H499R, M431V / M507L / I518V / G541E / A547G / V638I / L669H / S671N, M431V / M507L / I518V / L669H / S671N, A437S, K471Q / A478S, M507L / A547G / W610R, M507L / A547G / V638I / L669H / S671N, A547G, A547G / W610R / V638I / S671N, A547G / V638I / S668D, K581G, K581T, L642Q, L642S, L670N, T692Q, A750P, A753T, G820E, L871E, and C944G, these positions being numbered with reference to SEQ ID NO:2.,

[0009] The present invention provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 6. In some embodiments, acid alpha-glucosidase is 27, 27 / 944, 28, 29 / 218 / 240 / 668 / 700 / 744 / 869, 29 / 218 / 240 / 700 / 869, 29 / 240 / 596 / 668 / 700 / 744 / 869, 29 / 240 / 596 / 668 / 869, 29 / 478, 30, 36 / 106 / 150 / 218 / 527 / 750 / 883 / 894, 88, 106 / 112 / 150 / 218 / 414 / 5 27 / 793 / 883, 106 / 150 / 169 / 218 / 414 / 486 / 527 / 750 / 894, 106 / 150 / 169 / 218 / 414 / 486 / 527 / 894, 106 / 150 / 169 / 218 / 414 / 486 / 749 / 793 / 883 / 894, 106 / 150 / 169 / 218 / 414 / 486 / 750 / 793 / 883 / 894, 106 / 150 / 169 / 218 / 414 / 486 / 793 / 883, 106 / 15 0 / 169 / 218 / 414 / 486 / 894, 106 / 150 / 169 / 218 / 414 / 749 / 750 / 793 / 883, 106 / 150 / 169 / 218 / 414 / 749 / 793, 106 / 150 / 169 / 218 / 414 / 749 / 793 / 883, 106 / 150 / 169 / 218 / 486 / 527 / 749 / 793 / 894, 106 / 150 / 169 / 218 / 486 / 749 / 883, 106 / 150 / 169 / 218 / 486 / 883, 106 / 150 / 169 / 218 / 749 / 800, 106 / 150 / 169 / 414 / 486 / 749 / 750 / 883, 106 / 150 / 169 / 527 / 749 / 793 / 883, 106 / 150 / 169 / 749 / 793 / 883 / 894, 106 / 150 / 218 / 331 / 414 / 486 / 527 / 733 / 749 / 793, 106 / 150 / 218 / 414 / 486 / 642 / 750 / 793 / 883,106 / 150 / 218 / 414 / 486 / 750 / 793 / 894、106 / 150 / 218 / 414 / 527 / 749 / 750 / 883、106 / 150 / 218 / 414 / 527 / 749 / 793 / 883 / 894、106 / 150 / 218 / 414 / 749 / 750 / 793 / 883 / 894、106 / 150 / 218 / 414 / 749 / 793 / 883、106 / 150 / 218 / 486 / 527 / 749 / 894、106 / 150 / 218 / 486 / 793 / 883、106 / 150 / 218 / 527 / 749 / 750 / 793、106 / 150 / 218 / 527 / 793 / 894、106 / 150 / 218 / 749 / 750 / 793、106 / 150 / 218 / 793、106 / 150 / 218 / 793 / 894、106 / 150 / 245 / 793 / 883 / 894、106 / 150 / 414 / 749 / 750 / 793 / 894、106 / 150 / 414 / 749 / 793 / 894、106 / 150 / 486 / 527 / 750 / 793、106 / 150 / 486 / 749 / 793 / 883 / 894、106 / 150 / 749 / 793 / 883、106 / 169 / 185 / 218 / 414 / 749 / 750 / 793、106 / 191 / 280 / 402 / 414 / 444 / 727、106 / 191 / 414 / 444 / 522 / 928 / 944、106 / 191 / 414 / 489 / 928 / 944、106 / 280 / 402 / 414 / 444 / 489 / 727 / 944、107、109、109 / 842、110、113、135、137、138、148、150、150 / 169 / 218 / 414 / 527 / 793、150 / 218 / 414 / 486 / 749 / 750、150 / 218 / 414 / 486 / 750 / 793、150 / 218 / 414 / 486 / 750 / 793 / 883、150 / 218 / 414 / 749 / 750 / 793 / 894、150 / 218 / 414 / 749 / 793、150 / 218 / 527 / 749 / 793、150 / 218 / 749 / 750 / 793、150 / 218 / 749 / 793、150 / 414 / 486 / 527 / 750 / 894、150 / 414 / 486 / 749 / 750 / 793、150 / 486 / 750 / 883 / 894、169 / 486 / 750 / 793 / 883、180 / 275 / 402 / 518 / 547 / 610 / 638 / 669 / 671、180 / 402 / 431 / 507 / 547 / 610 / 669 / 671 / 793、180 / 402 / 507 / 547 / 610 / 671、191 / 280 / 402 / 414 / 444 / 465 / 842 / 928、191 / 280 / 402 / 414 / 444 / 489 / 500 / 944、191 / 280 / 414 / 444 / 489 / 500 / 522 / 842 / 928 / 944、191 / 280 / 414 / 444 / 489 / 522 / 727 / 944、191 / 280 / 414 / 489 / 842 / 928 / 944、191 / 280 / 414 / 944、191 / 414 / 522 / 842 / 944、196 / 402 / 431 / 547 / 610 / 638、218 / 668 / 700 / 869、224 / 402 / 507 / 518 / 547 / 638 / 668、247、269 / 275 / 431 / 518 / 547 / 638 / 668 / 669、274、275 / 281 / 402 / 431 / 507 / 518 / 610 / 668、275 / 281 / 402 / 431 / 518 / 547 / 610 / 669 / 671、275 / 281 / 402 / 507 / 518 / 547 / 638 / 669 / 671、275 / 281 / 402 / 518 / 547 / 610 / 638 / 671、275 / 281 / 402 / 518 / 547 / 610 / 668 / 669 / 887、275 / 281 / 402 / 547 / 610 / 638 / 669 / 671、275 / 281 / 431 / 518 / 547 / 638 / 669 / 671、275 / 281 / 507 / 547 / 669 / 671、275 / 281 / 610 / 638 / 668 / 669、275 / 281 / 671、275 / 377 / 402 / 507 / 518 / 669 / 671 / 715、275 / 402 / 431 / 507 / 547 / 671、275 / 402 / 431 / 518 / 610 / 638 / 669 / 671 / 922、275 / 402 / 507 / 547 / 610 / 638 / 668 / 669、275 / 402 / 507 / 547 / 610 / 638 / 669 / 671、275 / 402 / 507 / 547 / 610 / 671、275 / 402 / 547 / 610 / 638 / 669 / 671、275 / 402 / 547 / 638 / 669 / 671、275 / 402 / 638 / 669 / 671、275 / 431 / 507 / 518 / 547 / 668 / 669 / 671、275 / 431 / 507 / 518 / 610 / 669 / 671、275 / 431 / 507 / 547 / 610 / 638 / 671, 275 / 431 / 518 / 547 / 638 / 668, 275 / 431 / 518 / 610 / 638 / 669 / 671, 275 / 431 / 638, 275 / 507 / 518 / 547 / 610 / 638 / 668 / 669, 275 / 507 / 518 / 547 / 638 / 669 / 671, 275 / 507 / 547 / 610 / 638 / 669 / 671, 275 / 507 / 547 / 668 / 6 69 / 671, 275 / 518 / 671, 276, 278, 280 / 402 / 536 / 928, 281 / 402 / 507 / 518 / 547 / 610 / 638 / 669 / 671, 281 / 402 / 507 / 547 / 638 / 669 / 671, 281 / 402 / 518 / 547 / 610 / 638 / 668 / 669, 281 / 402 / 518 / 547 / 668, 281 / 431 / 507 / 518 / 547 / 610 / 638 / 668, 375, 402 / 431 / 518 / 547 / 610 / 668, 402 / 431 / 518 / 547 / 671, 402 / 431 / 518 / 610, 402 / 431 / 547 / 638 / 671, 403, 414, 418, 418 / 499, 421, 426, 431 / 507 / 518 / 541 / 547 / 638 / 669 / 671, 431 / 507 / 518 / 669 / 671, 437, 444, 455, 463, 471, 471 / 478, 476, 489, 507 / 5 The positions or sets of positions selected from 47 / 610, 507 / 547 / 638 / 669 / 671, 527, 547, 547 / 610 / 638 / 671, 547 / 638 / 668, 581, 610, 642, 668, 670, 692, 725 / 732, 750, 753, 786, 820, 862, 871, 895, 897, 934, and 944 include at least one substitution, where these positions are numbered with reference to Sequence ID No. 6. In some embodiments, acid alpha-glucosidase is 27P, 27P / 944W, 27R, 28P, 28R, 28S, 29Q / 218S / 240I / 668D / 700F / 744V / 869L, 29Q / 240I / 596P / 668D / 869L, 29Q / 240I / 596S / 668D / 700F / 744V / 869T, 29T / 478T, 29V / 218S / 240I / 700F / 869T, 30G, 30K, 30T,36R / 106P / 150S / 218S / 527D / 750P / 883H / 894R、88G、88S、106P / 112S / 150S / 218S / 414G / 527D / 793K / 883H、106P / 150S / 169S / 218S / 414G / 486E / 527D / 750P / 894R、106P / 150S / 169S / 218S / 414G / 486E / 527D / 894R、106P / 150S / 169S / 218S / 414G / 486E / 749E / 793K / 883H / 894R、106P / 150S / 169S / 218S / 414G / 486E / 750P / 793K / 883H / 894R、106P / 150S / 169S / 218S / 414G / 486E / 793K / 883H、106P / 150S / 169S / 218S / 414G / 486E / 894R、106P / 150S / 169S / 218S / 414G / 749E / 750P / 793K / 883H、106P / 150S / 169S / 218S / 414G / 749E / 793K、106P / 150S / 169S / 218S / 414G / 749E / 793K / 883H、106P / 150S / 169S / 218S / 486E / 527D / 749E / 793K / 894R、106P / 150S / 169S / 218S / 486E / 749E / 883H、106P / 150S / 169S / 218S / 486E / 883H、106P / 150S / 169S / 218S / 749E / 800A、106P / 150S / 169S / 414G / 486E / 749E / 750P / 883H、106P / 150S / 169S / 527D / 749E / 793K / 883H、106P / 150S / 169S / 749E / 793K / 883H / 894R、106P / 150S / 218S / 331A / 414G / 486E / 527D / 733E / 749E / 793K、106P / 150S / 218S / 414G / 486E / 642F / 750P / 793K / 883H、106P / 150S / 218S / 414G / 486E / 750P / 793K / 894R、106P / 150S / 218S / 414G / 527D / 749E / 750P / 883H、106P / 150S / 218S / 414G / 527D / 749E / 793K / 883H / 894G、106P / 150S / 218S / 414G / 749E / 750P / 793K / 883H / 894R、106P / 150S / 218S / 414G / 749E / 793K / 883H、106P / 150S / 218S / 486E / 527D / 749E / 894R、106P / 150S / 218S / 486E / 793K / 883H、106P / 150S / 218S / 527D / 749E / 750P / 793K、106P / 150S / 218S / 527D / 793K / 894G、106P / 150S / 218S / 749E / 7、 50P / 793K、106P / 150S / 218S / 793K、106P / 150S / 218S / 793K / 894R、106P / 150S / 245S / 793K / 883H / 894R、106P / 150S / 414G / 749E / 750P / 793K / 894R、106P / 150S / 414G / 749E / 793K / 894R、106P / 150S / 486E / 527D / 750P / 793K、106P / 150S / 486E / 749E / 793K / 883H / 894G、106P / 150S / 749E / 793K / 883H、106P / 169S / 185G / 218S / 414G / 749E / 750P / 793K、106P / 191R / 280D / 402A / 414G / 444P / 727P、106P / 191R / 414G / 444P / 522V / 928T / 944S、106P / 191R / 414G / 489D / 928T / 944S、106P / 280D / 402A / 414G / 444P / 489D / 727P / 944S、107G、107P、109G / 842E、109P、110G、110L、113S、135A、135Q、137P、138A、148G、148Y、150G、150S / 169S / 218S / 414G / 527D / 793K、150S / 218S / 414G / 486A / 750P / 793K、150S / 218S / 414G / 486E / 749E / 750P、150S / 218S / 414G / 486E / 750P / 793K / 883H、150S / 218S / 414G / 749E / 750P / 793K / 894R、150S / 218S / 414G / 749E / 793K、150S / 218S / 527D / 749E / 793K、150S / 218S / 749E / 750P / 793K、150S / 218S / 749E / 793K、150S / 414G / 486E / 527D / 750P / 894R、150S / 414G / 486E / 749E / 750P / 793K、150S / 486E / 750P / 883H / 894G、169S / 486E / 750P / 793K / 883H、180H / 275M / 402A / 518V / 547G / 610R / 638I / 669H / 671N、180H / 402A / 431V / 507L / 547G / 610R / 669H / 671N / 793G、180H / 402A / 507L / 547G / 610R / 671N、191R / 280D / 402A / 414G / 444P / 465E / 842S / 928T、191R / 280D / 402A / 414G / 444P / 489D / 500A / 944S、191R / 280D / 414G / 444P / 489D / 500A / 522V / 842S / 928T / 944S、191R / 280D / 414G / 444P / 489D / 522V / 727P / 944S、191R / 280D / 414G / 489D / 842S / 928T / 944S、191R / 280D / 414G / 944S、191R / 414G / 522V / 842S / 944S、196V / 402A / 431V / 547G / 610R / 638I、218S / 668D / 700F / 869T、224F / 402A / 507L / 518V / 547G / 638I / 668D、247R、269N / 275M / 431V / 518V / 547G / 638I / 668D / 669H、274G、275M / 281V / 402A / 431V / 507L / 518V / 610R / 668D、275M / 281V / 402A / 507L / 518V / 547G / 638I / 669H / 671N、275M / 281V / 402A / 518V / 547G / 610R / 638I / 671N、275M / 281V / 402A / 518V / 547G / 610R / 668D / 669H / 887D、275M / 281V / 402A / 547G / 610R / 638I / 669H / 671N、275M / 281V / 507L / 547G / 669H / 671N、275M / 281V / 610R / 638I / 668D / 669H、275M / 402A / 431V / 507L / 547G / 671N、275M / 402A / 507L / 547G / 610R / 671N、275M / 402A / 547G / 638I / 669H / 671N、275M / 431V / 518V / 547G / 638I / 668D、275M / 431V / 518V / 610R / 638I / 669H / 671N、275M / 431V / 638I、275M / 507L / 547G / 668D / 669H / 671N、275V / 281V / 402A / 431V / 518V / 547G / 610R / 669H / 671N、275V / 281V / 431V / 518V / 547G / 638I / 669H / 671N、275V / 281V / 671N、275V / 377K / 402A / 507L / 518V / 669H / 671N / 715G、275V / 402A / 431V / 518V / 610R / 638I / 669H / 671N / 922L、275V / 402A / 507L / 547G / 610R / 638I / 668D / 669H、275V / 402A / 507L / 547G / 610R / 638I / 669H / 671N、275V / 402A / 547G / 610R / 638I / 669H / 671N、275V / 402A / 638I / 669H / 671N、275V / 431V / 507L / 518V / 547G / 668D / 669H / 671N、275V / 431V / 507L / 518V / 610R / 669H / 671N、275V / 431V / 507L / 547G / 610R / 638I / 671N、275V / 507L / 518V / 547G / 610R / 638I / 668D / 669H、275V / 507L / 518V / 547G / 638I / 669H / 671N、275V / 507L / 547G / 610R / 638I / 669H / 671N、275V / 518V / 671N、276F、276Y、278A、278G、280D / 402A / 536I / 928T、281V / 402A / 507L / 518V / 547G / 610R / 638I / 669H / 671N、281V / 402A / 507L / 547G / 638I / 669H / 671N、281V / 402A / 518V / 547G / 610R / 638I / 668D / 669H、281V / 402A / 518V / 547G / 668D、281V / 431V / 507L / 518V / 547G / 610R / 638I / 668D、375E、402A / 431V / 518V / 547G / 610R / 668D、402A / 431V / 518V / 547G / 671N、402A / 431V / 518V / 610R、402A / 431V / 547G / 638I / 671N、403W、414P、418E / 499R、418R、421S、426R、431V / 507L / 518V / 541E / 547G / 638I / 669H / 671N、431V / 507L / 518V / 669H / 671N、437S、444T、455V、463A、471Q / 478S、471S、476A、476H、489R、507L / 547G / 610R、507L / 547G / 638I / 669H / 671N、527R、547G、547G / 610R / 638I / 671N、547G / 638I / 668D、581G、581T、610A、The substitution includes at least one substitution in a position or set of positions selected from 610G, 610S, 642M, 642Q, 642S, 668H, 670N, 692Q, 725N / 732I, 750P, 753T, 786P, 786Y, 820E, 862G, 871E, 895R, 897V, 934R, 944G, and 944R, where these positions are numbered with reference to Sequence ID No. 6. In some embodiments, the acid alpha-glucosidase is F27P, F27P / C944W, F27R, L28P, L28R, L28S, L29Q / L218S / L240I / S668D / H700F / I744V / I869L, L29Q / L240I / A596P / S668D / I869L, L29Q / L240I / A596S / S668D / H700F / I744V / I869T, L29T / A478T, L29V / L218S / L2 40I / H700F / I869T, V30G, V30K, V30T, G36R / K106P / T150S / L218S / N527D / A750P / R883H / Q894R, K88G, K88S, K106P / A112 S / T150S / L218S / R414G / N527D / E793K / R883H, K106P / T150S / N169S / L218S / R414G / T486E / N527D / A750P / Q894R, K106P / T 150S / N169S / L218S / R414G / T486E / N527D / Q894R, K106P / T150S / N169S / L218S / R414G / T486E / Q749E / E793K / R883H / Q89 4R, K106P / T150S / N169S / L218S / R414G / T486E / A750P / E793K / R883H / Q894R, K106P / T150S / N169S / L218S / R414G / T486E / E793K / R883H, K106P / T150S / N169S / L218S / R414G / T486E / Q894R, K106P / T150S / N169S / L218S / R414G / Q749E / A750P / E79 3K / R883H, K106P / T150S / N169S / L218S / R414G / Q749E / E793K, K106P / T150S / N169S / L218S / R414G / Q749E / E793K / R883H,K106P / T150S / N169S / L218S / T486E / N527D / Q749E / E793K / Q894R、K106P / T150S / N169S / L218S / T486E / Q749E / R883H、K106P / T150S / N169S / L218S / T486E / R883H、K106P / T150S / N169S / L218S / Q749E / P800A、K106P / T150S / N169S / R414G / T486E / Q749E / A750P / R883H、K106P / T150S / N169S / N527D / Q749E / E793K / R883H、K106P / T150S / N169S / Q749E / E793K / R883H / Q894R、K106P / T150S / L218S / V331A / R414G / T486E / N527D / D733E / Q749E / E793K、K106P / T150S / L218S / R414G / T486E / L642F / A750P / E793K / R883H、K106P / T150S / L218S / R414G / T486E / A750P / E793K / Q894R、K106P / T150S / L218S / R414G / N527D / Q749E / A750P / R883H、K106P / T150S / L218S / R414G / N527D / Q749E / E793K / R883H / Q894G、K106P / T150S / L218S / R414G / Q749E / A750P / E793K / R883H / Q894R、K106P / T150S / L218S / R414G / Q749E / E793K / R883H、K106P / T150S / L218S / T486E / N527D / Q749E / Q894R、K106P / T150S / L218S / T486E / E793K / R883H、K106P / T150S / L218S / N527D / Q749E / A750P / E793K、K106P / T150S / L218S / N527D / E793K / Q894G、K106P / T150S / L218S / Q749E / A750P / E793K、K106P / T150S / L218S / E793K、K106P / T150S / L218S / E793K / Q894R、K106P / T150S / P245S / E793K / R883H / Q894R、K106P / T150S / R414G / Q749E / A750P / E793K / Q894R、K106P / T150S / R414G / Q749E / E793K / Q894R、K106P / T150S / T486E / N527D / A750P / E793K、K106P / T150S / T486E / Q749E / E793K / R883H / Q894G、K106P / T150S / Q749E / E793K / R883H、K106P / N169S / V185G / L218S / R414G / Q749E / A750P / E793K、K106P / H19、 1R / G280D / S402A / R414G / A444P / S727P、K106P / H191R / R414G / A444P / E522V / D928T / C944S、K106P / H191R / R414G / A489D / D928T / C944S、K106P / G280D / S402A / R414G / A444P / A489D / S727P / C944S、Q107G、Q107P、L109G / G842E、L109P、Q110G、Q110L、Q113S、S135A、S135Q、E137P、M138A、T148G、T148Y、T150G、T150S / N169S / L218S / R414G / N527D / E793K、T150S / L218S / R414G / T486A / A750P / E793K、T150S / L218S / R414G / T486E / Q749E / A750P、T150S / L218S / R414G / T486E / A750P / E793K / R883H、T150S / L218S / R414G / Q749E / A750P / E793K / Q894R、T150S / L218S / R414G / Q749E / E793K、T150S / L218S / N527D / Q749E / E793K、T150S / L218S / Q749E / A750P / E793K、T150S / L218S / Q749E / E793K、T150S / R414G / T486E / N527D / A750P / Q894R、T150S / R414G / T486E / Q749E / A750P / E793K、T150S / T486E / A750P / R883H / Q894G、N169S / T486E / A750P / E793K / R883H、N180H / L275M / S402A / I518V / A547G / W610R / V638I / L669H / S671N、N180H / S402A / M431V / M507L / A547G / W610R / L669H / S671N / E793G、N180H / S402A / M507L / A547G / W610R / S671N、H191R / G280D / S402A / R414G / A444P / G465E / G842S / D928T、H191R / G280D / S402A / R414G / A444P / A489D / D500A / C944S、H191R / G280D / R414G / A444P / A489D / D500A / E522V / G842S / D928T / C944S、H191R / G280D / R414G / A444P / A489D / E522V / S727P / C944S、H191R / G280D / R414G / A489D / G842S / D928T / C944S、H191R / G280D / R414G / C944S、H191R / R414G / E522V / G842S / C944S、A196V / S402A / M431V / A547G / W610R / V638I、L218S / S668D / H700F / I869T、L224F / S402A / M507L / I518V / A547G / V638I / S668D、Q247R、T269N / L275M / M431V / I518V / A547G / V638I / S668D / L669H、D274G、L275M / A281V / S402A / M431V / M507L / I518V / W610R / S668D、L275M / A281V / S402A / M507L / I518V / A547G / V638I / L669H / S671N、L275M / A281V / S402A / I518V / A547G / W610R / V638I / S671N、L275M / A281V / S402A / I518V / A547G / W610R / S668D / L669H / E887D、L275M / A281V / S402A / A547G / W610R / V638I / L669H / S671N、L275M / A281V / M507L / A547G / L669H / S671N、L275M / A281V / W610R / V638I / S668D / L669H、L275M / S402A / M431V / M507L / A547G / S671N、L275M / S402A / M507L / A547G / W610R / S671N、L275M / S402A / A547G / V638I / L669H / S671N、L275M / M431V / I518V / A547G / V638I / S668D、L275M / M431V / I518V / W610R / V638I / L669H / S671N、L275M / M431V / V638I、L275M / M507L / A547G / S668D / L669H / S671N、L275V / A281V / S402A / M431V / I518V / A547G / W610R / L669H / S671N、L275V / A281V / M431V / I518V / A547G / V638I / L669H / S671N、L275V / A281V / S671N、L275V / R377K / S402A / M507L / I518V / L669H / S671N / V715G、L275V / S402A / M431V / I518V / W610R / V638I / L669H / S671N / P922L、L275V / S402A / M507L / A547G / W610R / V638I / S668D / L669H、L275V / S402A / M507L / A547G / W610R / V638I / L669H / S671N、L275V / S402A / A547G / W610R / V638I / L669H / S671N、L275V / S402A / V638I / L669H / S671N、L275V / M431V / M507L / I518V / A547G / S668D / L669H / S671N、L275V / M431V / M507L / I518V / W610R / L669H / S671N、L275V / M431V / M507L / A547G / W610R / V638I / S671N、L275V / M507L / I518V / A547G / W610R / V638I / S668D / L669H、L275V / M507L / I518V / A547G / V638I / L669H / S671N、L275V / M507L / A547G / W610R / V638I / L669H / S671N、L275V / I518V / S671N、A276F、A276Y、T278A、T278G、G280D / S402A / V536I / D928T、A281V / S402A / M507L / I518V / A547G / W610R / V638I / L669H / S671N、A281V / S402A / M507L / A547G / V638I / L669H / S671N、A281V / S402A / I518V / A547G / W610R / V638I / S668D / L669H、A281V / S402A / I518V / A547G / S668D、A281V / M431V / M507L / I518V / A547G / W610R / V638I / S668D、I375E、S402A / M431V / I518V / A547G / W610R / S668D、S402A / M431V / I518V / A547G / S671N、S402A / M431V / I518V / W610R、S402A / M431V / A547G / V638I / S671N、R403W、R414P、A418E / H499R、A418R、Q421S、G426R、M431V / M507L / I518V / G541E / A547G / V638I / L669H / S671N, M431V / M507L / I518V / L669H / S671N, A437S, A444T, R455V, E463A, K471Q / A478S , K471S, S476A, S476H, A489R, M507L / A547G / W610R, M507L / A547G / V638I / L669H / S671N, N527R, A547G, A547G / W610R / V638I / S671N, A547 The system includes at least one substitution in a position or set of positions selected from G / V638I / S668D, K581G, K581T, W610A, W610G, W610S, L642M, L642Q, L642S, S668H, L670N, T692Q, K725N / V732I, A750P, A753T, R786P, R786Y, G820E, R862G, L871E, K895R, T897V, L934R, C944G, and C944R, where these positions are numbered with reference to Sequence ID No. 6.

[0010] The present invention provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 8. In some embodiments, acid alpha-glucosidase is 4, 27, 27 / 28 / 489, 27 / 418 / 478, 28, 28 / 29, 28 / 29 / 113 / 135 / 138, 28 / 29 / 113 / 135 / 418, 28 / 29 / 135, 28 / 29 / 418, 29 / 113 / 126 / 135 / 193, 29 / 113 / 135, 29 / 113 / 135 / 455, 29 / 113 / 138, 29 / 148, 29 / 478, 106, 106 / 138 / 218 / 431 / 671 / 749, 106 / 218 / 281, 106 / 218 / 455, 106 / 218 / 455 / 507 / 749, 106 / 489 / 671, 106 / 638, 106 / 671 / 934, 113, 113 / 135 / 418, 113 / 418 / 455 / 478 / 581, 113 / 418 / 478 / 489 / 581, 135, 135 / 148 / 150 / 418, 135 / 478 / 489 / 581, 135 / 489, 135 / 944, 138 / 218 / 668 / 671, 138 / 218 / 749 / 934, 138 / 671 / 749 / 934, 157, 218, 218 / 281, 218 / 281 / 431, 218 / 281 / 671, 218 / 431, 218 / 431 / 489 / 507 / 749 / 934, 218 / 455, 218 / 507 / 749, 218 / 507 / 934, 218 / 638 / 671, 218 / 749, 281 The system includes at least one substitution in a position or set of positions selected from / 431 / 489 / 668, 345 / 934, 418, 418 / 489, 431 / 668 / 671, 489 / 638 / 934, 489 / 671 / 934, 489 / 749, 489 / 934, 507 / 668, 507 / 671 / 934, 671 / 749, 671 / 934, and 749 / 784, where these positions are numbered with reference to Sequence ID No. 8. In some embodiments,Acid alpha-glucosidases include 4H, 27P / 28S / 489R, 27P / 418E / 478T, 27R, 28S, 28S / 29T, 28S / 29T / 113S / 135Q / 138A, 28S / 29T / 113S / 135Q / 418E, 28S / 29T / 135Q, 28S / 29T / 418E, 29T / 113S / 126Q / 135Q / 193Q, 29T / 113S / 135Q, 29T / 113S / 135Q / 455V, 29T / 113S / 138A, 29T / 148G, 29T / 478T, 106P, and 106P / 1 38A / 218S / 431V / 671N / 749E, 106P / 218S / 281V, 106P / 218S / 455V, 106P / 218S / 455V / 507L / 749E, 106P / 489R / 671N, 106P / 638I, 106P / 671N / 934 R, 113S, 113S / 135Q / 418E, 113S / 418E / 455V / 478T / 581T, 113S / 418E / 47 8T / 489R / 581T, 135P / 944Y, 135Q, 135Q / 148G / 150G / 418E, 135Q / 478T / 4 89R / 581T, 135Q / 489R, 138A / 218S / 668D / 671N, 138A / 218S / 749E / 934R , 138A / 671N / 749E / 934R, 157M, 218S, 218S / 281V, 218S / 281V / 431V, 218 S / 281V / 671N, 218S / 431V, 218S / 431V / 489R / 507L / 749E / 934R, 218S / 4 55V, 218S / 507L / 749E, 218S / 507L / 934R, 218S / 638I / 671N, 218S / 749E, The acidic alpha-glucosidase includes at least one substitution or set of substitutions at one or more positions selected from 281V / 431V / 489R / 668D, 345K / 934R, 418E, 418E / 489R, 431V / 668D / 671N, 489R / 638I / 934R, 489R / 671N / 934R, 489R / 749E, 489R / 934R, 507L / 668D, 507L / 671N / 934R, 671N / 749E, 671N / 934R, and 749E / 784T, where these positions are numbered with reference to Sequence ID No. 8. In some embodiments, the acidic alpha-glucosidase includes P4H, F27P / L28S / A489R, F27P / A418E / A478T,F27R、L28S、L28S / L29T、L28S / L29T / Q113S / S135Q / M138A、L28S / L29T / Q113S / S135Q / A418E、L28S / L29T / S135Q、L28S / L29T / A418E、L29T / Q113S / P126Q / S135Q / H193Q、L29T / Q113S / S135Q、L29T / Q113S / S135Q / R455V、L29T / Q113S / M138A、L29T / T148G、L29T / A478T、K106P、K106P / M138A / L218S / M431V / S671N / Q749E、K106P / L218S / A281V、K106P / L218S / R455V、K106P / L218S / R455V / M507L / Q749E、K106P / A489R / S671N、K106P / V638I、K106P / S671N / L934R、Q113S、Q113S / S135Q / A418E、Q113S / A418E / R455V / A478T / K581T、Q113S / A418E / A478T / A489R / K581T、S135P / C944Y、S135Q、S135Q / T148G / S150G / A418E、S135Q / A478T / A489R / K581T、S135Q / A489R、M138A / L218S / S668D / S671N、M138A / L218S / Q749E / L934R、M138A / S671N / Q749E / L934R、L157M、L218S、L218S / A281V、L218S / A281V / M431V、L218S / A281V / S671N、L218S / M431V、L218S / M431V / A489R / M507L / Q749E / L934R、L218S / R455V、L218S / M507L / Q749E、L218S / M507L / L934R、L218S / V638I / S671N、L218S / Q749E、A281V / M431V / A489R / S668D、Q345K / L934R、A418E、A418E / A489R、M431V / S668D / S671N、A489R / V638I / L934R、A489R / S671N / L934R、A489R / Q749E、A489R / L934R、M507L / S668D、M507L / S671N / L934R、S671N / Q749E、S671N / L934R、and include at least one substitution or set of substitutions at one or more positions selected from Q749E / A784, where these positions are numbered with reference to Sequence ID 8. In some embodiments, acid alpha-glucosidase is 27, 27 / 418 / 478, 28, 28 / 29, 28 / 29 / 113 / 135 / 138, 28 / 29 / 113 / 135 / 418, 28 / 29 / 135, 28 / 29 / 418, 29 / 113 / 126 / 135 / 193, 29 / 113 / 135, 29 / 113 / 135 / 455, 29 / 113 / 138, 29 / 148, 29 / 478, 106, 106 / 138 / 218 / 431 / 671 / 749, 106 / 489 / 671, 106 / 638, 113 / 135 The system includes at least one substitution or set of substitutions at one or more positions selected from / 418, 113 / 418 / 455 / 478 / 581, 113 / 418 / 478 / 489 / 581, 135, 135 / 148 / 150 / 418, 135 / 478 / 489 / 581, 135 / 944, 138 / 218 / 668 / 671, 157, 218 / 638 / 671, 418, 418 / 489, 431 / 668 / 671, 507 / 668, and 671 / 749, where these positions are numbered with reference to Sequence ID No. 8. In some embodiments, the acidic alpha-glucosidase is 27P / 418E / 478T, 27R, 28S, 28S / 29T, 28S / 29T / 113S / 135Q / 138A, 28S / 29T / 113S / 135Q / 418E, 28S / 29T / 135Q, 28S / 29T / 418E, 29T / 113S / 126Q / 135Q / 193Q, 29T / 113S / 135Q, 29T / 113S / 135Q / 455V, 29T / 113S / 138A, 29T / 148G, 29T / 478T, 106P, 106P / 138A / 218S / 431V / 671N / 749E, 106P / 489R / 671N, 106P / 638I, 113S / 135Q / 418E, 113S / 418E / 455V / 478T / 581T, 113S / 418E / 478T / 489R / 58 1T, 135P / 944Y, 135Q, 135Q / 148G / 150G / 418E, 135Q / 478T / 489R / 581T, 138A / 218S / 668D / 671N, 157M, 218S / 638I / 671N, 418E, 418E / 489R,The acidic alpha-glucosidase includes at least one substitution or set of substitutions at one or more positions selected from 431V / 668D / 671N, 507L / 668D, and 671N / 749E, where these positions are numbered with reference to Sequence ID No. 8. In some embodiments, the acidic alpha-glucosidase includes F27P / A418E / A478T, F27R, L28S, L28S / L29T, L28S / L29T / Q113S / S135Q / M138A, L28S / L29T / Q113S / S135Q / A418E, L28S / L29T / S135Q, L28S / L29T / A418E, L29T / Q113S / P 126Q / S135Q / H193Q, L29T / Q113S / S135Q, L29T / Q113S / S135Q / R455V, L29T / Q113S / M138A, L29T / T1 48G, L29T / A478T, K106P, K106P / M138A / L218S / M431V / S671N / Q749E, K106P / A489R / S671N, K106P / V 638I, Q113S / S135Q / A418E, Q113S / A418E / R455V / A478T / K581T, Q113S / A418E / A478T / A489R / K581 T, S135P / C944Y, S135Q, S135Q / T148G / S150G / A418E, S135Q / A478T / A489R / K581T, M138A / L218S / S6 It includes at least one substitution or set of substitutions at one or more positions selected from 68D / S671N, L157M, L218S / V638I / S671N, A418E, A418E / A489R, M431V / S668D / S671N, M507L / S668D, and S671N / Q749E, where these positions are numbered with reference to Sequence ID 8.

[0011] The present invention also provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 12. In some embodiments, acid alpha-glucosidase is 4, 27, 27 / 28 / 489, 27 / 418 / 478, 28, 28 / 29, 28 / 29 / 113 / 135 / 138, 28 / 29 / 113 / 135 / 418, 28 / 29 / 135, 28 / 29 / 418, 29 / 113 / 126 / 135 / 193, 29 / 113 / 135, 29 / 113 / 135 / 455, 29 / 113 / 138, 29 / 148, 29 / 478, 106, 106 / 138 / 218 / 431 / 671 / 749, 106 / 218 / 281, 106 / 218 / 455, 106 / 218 / 455 / 507 / 749, 106 / 489 / 671, 106 / 638, 106 / 671 / 934, 113, 113 / 135 / 418, 113 / 418 / 455 / 478 / 581, 113 / 418 / 478 / 489 / 581, 135, 135 / 148 / 15 0 / 418, 135 / 478 / 489 / 581, 135 / 489, 135 / 944, 138 / 218 / 668 / 671, 138 / 218 / 749 / 934, 138 / 671 / 749 / 934, 157, 218, 218 / 281 / 431, 218 / 281 / 671, 218 / 431, 218 / 455, 218 / 507 / 749, 218 / 638 / 671, 218 / 749, 281 / 431 / 489 / 668 ,345 / 934,418,418 / 489,431 / 668 / 671,489 / 638 / 934,489 / 671 / 934,489 / 749,489 / 934,507 / 668,507 / 671 / 934,671 / 749,671 / 934, and 749 / 784, the substitution includes at least one substitution in a position or set of positions selected from these, the positions being numbered with reference to Sequence ID No. 12. In some embodiments, the acid alpha-glucosidase is 4H, 27P / 28S / 489R, 27P / 418E / 478T, 27R,28S, 28S / 29T, 28S / 29T / 113S / 135Q / 138A, 28S / 29T / 113S / 135Q / 418E, 28S / 29T / 135Q, 28S / 29T / 418E, 29T / 113S / 126Q / 135Q / 193Q, 29T / 113S / 135Q, 29T / 113S / 135Q / 455V, 29T / 113S / 138A, 29T / 148G, 29T / 478T, 106P, 106P / 138A / 218S / 431V / 671N / 749 E, 106P / 218S / 281V, 106P / 218S / 455V, 106P / 218S / 455V / 507L / 749E, 106P / 489R / 671N, 106P / 638I, 106P / 671N / 934R, 113S, 1 13S / 135Q / 418E, 113S / 418E / 455V / 478T / 581T, 113S / 418E / 478T / 489R / 581T, 135P / 944Y, 135Q, 135Q / 148G / 150G / 418E, 135Q / 478T / 489R / 581T, 135Q / 489R, 138A / 218S / 668D / 671N, 138A / 218S / 749E / 934R, 138A / 671N / 749E / 934R, 157M, 218S, 218S / 281 V / 431V, 218S / 281V / 671N, 218S / 431V, 218S / 455V, 218S / 507L / 749E, 218S / 638I / 671N, 218S / 749E, 281V / 431V / 489R / 668D, 34 The substitution includes at least one substitution in a position or set of positions selected from 5K / 934R, 418E, 418E / 489R, 431V / 668D / 671N, 489R / 638I / 934R, 489R / 671N / 934R, 489R / 749E, 489R / 934R, 507L / 668D, 507L / 671N / 934R, 671N / 749E, 671N / 934R, and 749E / 784T, where these positions are numbered with reference to Sequence ID No. 12. In some embodiments, the acidic alpha-glucosidase is P4H, F27P / L28S / A489R, F27P / A418E / A478T, F27R, L28S, L28S / L29T, L28S / L29T / Q113S / S135Q / M138A, L28S / L29T / Q113S / S135Q / A418E, L28S / L29T / S135Q, L28S / L29T / A418E,L29T / Q113S / P126Q / S135Q / H193Q, L29T / Q113S / S135Q, L29T / Q113S / S135Q / R455V, L29T / Q113S / M138A, L29T / T148G, L29T / A478T, K1 06P, K106P / M138A / L218S / M431V / S671N / Q749E, K106P / L218S / A281V, K106P / L218S / R455V, K106P / L218S / R455V / M507L / Q749E, K106 P / A489R / S671N, K106P / V638I, K106P / S671N / L934R, Q113S, Q113S / S135Q / A418E, Q113S / A418E / R455V / A478T / K581T, Q113S / A418E / A478T / A489R / K581T, S135P / C944Y, S135Q, S135Q / T148G / S150G / A418E, S135Q / A478T / A489R / K581T, S135Q / A489R, M138A / L218S / S6 68D / S671N, M138A / L218S / Q749E / L934R, M138A / S671N / Q749E / L934R, L157M, L218S, L218S / A281V / M431V, L218S / A281V / S671N, L218 S / M431V, L218S / R455V, L218S / M507L / Q749E, L218S / V638I / S671N, L218S / Q749E, A281V / M431V / A489R / S668D, Q345K / L934R, A418E, The system includes at least one substitution in a position or set of positions selected from A418E / A489R, M431V / S668D / S671N, A489R / V638I / L934R, A489R / S671N / L934R, A489R / Q749E, A489R / L934R, M507L / S668D, M507L / S671N / L934R, S671N / Q749E, S671N / L934R, and Q749E / A784T, where these positions are numbered with reference to Sequence ID No. 12.

[0012] The present invention provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 14. The present invention provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 16. In some embodiments, acid alpha-glucosidase is 22, 24, 27, 27 / 165, 30, 33, 34, 37 / 62, 37 / 62 / 79 / 196 / 696 / 862, 37 / 62 / 523, 37 / 62 / 523 / 793, 37 / 64 / 66 / 79 / 154 / 523 / 681 / 793 / 862, 37 / 79 / 15 4 / 793, 37 / 196, 37 / 528 / 696 / 793, 37 / 528 / 790, 37 / 528 / 790 / 793 / 862, 37 / 790 / 793, 39, 39 / 58 / 489 / 725 / 830 / 842 / 930 / 944, 39 / 70 / 109 / 830 / 842, 39 / 70 / 489 / 612, 39 / 70 / 725 , 39 / 267, 39 / 267 / 489 / 522 / 612 / 830 / 842, 39 / 267 / 489 / 830 / 944, 39 / 489 / 500 / 612, 39 / 500 / 612, 40, 44 / 157, 47, 49, 50, 55, 60 / 500 / 612, 62 / 79 / 154 / 862, 62 / 79 / 196 / 681 / 8 62, 62 / 79 / 523 / 528 / 790, 62 / 79 / 790 / 793, 62 / 79 / 862, 62 / 92, 62 / 92 / 790 / 793, 62 / 106 / 523 / 528 / 696 / 793 / 862, 62 / 154 / 696 / 793 / 862, 62 / 793 / 862, 68, 70, 70 / 267 / 725 / 944,70 / 267 / 930 / 944, 70 / 489 / 930, 70 / 725 / 830 / 860 / 930 / 944, 77, 79 / 154 / 681, 79 / 154 / 793 / 862, 79 / 862, 89, 97, 106 / 154, 107, 109, 109 / 522 / 612 / 725, 109 / 522 / 8 30 / 944, 109 / 612, 118, 149, 157, 158, 178, 179, 196 / 528 / 681 / 790 / 793, 207, 208, 217, 267 / 489 / 500 / 725 / 830 / 930, 267 / 522 / 725, 352, 385, 424, 448, 463, 489 / 8 The sequence includes at least one substitution in a position or set of positions selected from 30 / 944, 500, 500 / 612 / 830 / 860, 500 / 860 / 930, 500 / 930 / 944, 522 / 725, 523, 523 / 790 / 793, 528 / 681, 528 / 793, 528 / 862, 672, 673, 725, 734, 740, 753, 774, 778, 793, 830, 844, 862, 875, 880, 892, 902, 922, 925, 930, 932, 934, 938, and 944, where these positions are numbered with reference to sequence numbers 14 and / or 16. In some embodiments, acid alpha-glucosidase is 22R, 24E, 24R, 24W, 27A, 27G, 27G / 165I, 27K, 27R, 27S, 27V, 27W, 30D, 30L, 33G, 33P, 34D, 34M, 34T, 37F / 62E, 37F / 62E / 79S / 196T / 696S / 862Q, 37F / 62E / 523N, 37F / 62E / 523N / 793K, 37F / 64Q / 66G / 79S / 154R / 523N / 681Q / 793K / 862Q, 37F / 79S / 154R / 79 3K, 37F / 196T, 37F / 528S / 696S / 793K, 37F / 528S / 790V, 37F / 528S / 790V / 793K / 862Q, 37F / 790V / 793K, 39D, 39H, 39Q, 39Q / 58L / 489D / 725E / 83 0K / 842S / 930P / 944S, 39Q / 70A / 109P / 830K / 842S, 39Q / 70A / 489D / 612D, 39Q / 70A / 725E, 39Q / 267K, 39Q / 267K / 489D / 522V / 612D / 830K / 842S,39Q / 267K / 489D / 830K / 944S、39Q / 489D / 500A / 612D、39Q / 500A / 612D、40W、44I / 157V、47G、47R、49A、49G、50G、50L、50V、55C、55L、60V / 500A / 612D、62E / 79S / 154R / 862Q、62E / 79S / 196T / 681Q / 862Q、62E / 79S / 523N / 528S / 790V、62E / 79S / 790V / 793K、62E / 79S / 862Q、62E / 92R、62E / 92R / 790V / 793K、62E / 106R / 523N / 528S / 696S / 793K / 862Q、62E / 154R / 696S / 793K / 862Q、62E / 793K / 862Q、68N、68S、68W、70A / 267K / 725E / 944S、70A / 267K / 930P / 944S、70A / 489D / 930P、70A / 725E / 830K / 860F / 930P / 944S、70Q、77W、79S / 154R / 681Q、79S / 154R / 793K / 862Q、79S / 862Q、89R、97D、97G、106R / 154R、107G、109D、109P / 522V / 612D / 725E、109P / 522V / 830K / 944S、109P / 612D、118F、149R、157Q、158E、158F、178G、178V、179L、196T / 528S / 681Q / 790V / 793K、207R、207Y、208G、208I、217A、217D、267K / 489D / 500A / 725E / 830K / 930P、267K / 522V / 725E、352K、352V、385G、424K、448L、463A、489D / 830K / 944S、500A、500A / 612D / 830K / 860F、500A / 860F / 930P、500A / 930P / 944S、522V / 725E、523N、523N / 790V / 793K、528S / 681Q、528S / 793K、528S / 862Q、672E、672K、673N、673R、725F、725V、734K、740G、740Q、753S、774G、774S、778Q、793K、830V、844R、862Q、875D、880R、892L、902L、922E、925A、925W、930P、932A、934F、938A、938P、944R、and include at least one substitution or set of substitutions at one or more positions selected from 944S, where these positions are numbered with reference to sequence numbers 14 and / or 16. In some embodiments, acid alpha-glucosidase is I22R, L24E, L24R, L24W, F27A, F27G, F27G / M165I, F27K, F27R, F27S, F27V, F27W, V30D, V30L, E33G, E33P, L34D, L34M, L34T, S37F / A62E, S37F / A62E / N79S / A196T / A696S / R862Q, S37F / A62E / D523N, S37F / A62E / D523N / E793K, S37F / P64Q / R66G / N79S / K154R / D523N / E681Q / E793K / R862Q, S37F / N79S / K154R / E793K, S37F / A196T, S37F / N528S / A696S / E793K, S37F / N528S / I790V, S37 F / N528S / I790V / E793K / R862Q, S37F / I790V / E793K, P39D, P39H, P39Q, P39Q / R58L / A489D / K725E / Q830K / G842S / C930P / C944S, P39Q / V70 A / L109P / Q830K / G842S, P39Q / V70A / A489D / S612D, P39Q / V70A / K725E, P39Q / R267K, P39Q / R267K / A489D / E522V / S612D / Q830K / G842S, P 39Q / R267K / A489D / Q830K / C944S, P39Q / A489D / D500A / S612D, P39Q / D500A / S612D, V40W, T44I / L157V, A47G, A47R, Q49A, Q49G, Q50G, Q50 L, Q50V, P55C, P55L, A60V / D500A / S612D, A62E / N79S / K154R / R862Q, A62E / N79S / A196T / E681Q / R862Q, A62E / N79S / D523N / N528S / I790V , A62E / N79S / I790V / E793K, A62E / N79S / R862Q, A62E / Q92R, A62E / Q92R / I790V / E793K, A62E / K106R / D523N / N528S / A696S / E793K / R862Q,A62E / K154R / A696S / E793K / R862Q, A62E / E793K / R862Q, R68N, R68S, R68 W, V70A / R267K / K725E / C944S, V70A / R267K / C930P / C944S, V70A / A489D / C930P, V70A / K725E / Q830K / L860F / C930P / C944S, V70Q, P77W, N79S / K15 4R / E681Q, N79S / K154R / E793K / R862Q, N79S / R862Q, A89R, A97D, A97G, K 106R / K154R, Q107G, L109D, L109P / E522V / S612D / K725E, L109P / E522V / Q830K / C944S, L109P / S612D, W118F, P149R, L157Q, T158E, T158F, P178G , P178V, A179L, A196T / N528S / E681Q / I790V / E793K, E207R, E207Y, E208 G, E208I, Q217A, Q217D, R267K / A489D / D500A / K725E / Q830K / C930P, R26 7K / E522V / K725E, Y352K, Y352V, R385G, H424K, R448L, E463A, A489D / Q8 30K / C944S, D500A, D500A / S612D / Q830K / L860F, D500A / L860F / C930P, D 500A / C930P / C944S, E522V / K725E, D523N, D523N / I790V / E793K, N528S / E681Q, N528S / E793K, N528S / R862Q, L672E, L672K, P673N, P673R, K725F , includes at least one substitution or set of substitutions at one or more positions selected from K725V, H734K, E740G, E740Q, A753S, A774G, A774S, L778Q, E793K, Q830V, E844R, R862Q, N875D, E880R, Q892L, A902L, P922E, K925A, K925W, C930P, S932A, L934F, Q938A, Q938P, C944R, and C944S, where these positions are numbered with reference to sequence numbers 14 and / or 16.

[0013] The present invention also provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to Sequence ID No. 18, the position of which is numbered with reference to Sequence ID No. 18. In some embodiments, acid alpha-glucosidase is 22, 24, 27, 27 / 165, 30, 33, 34, 37 / 62, 37 / 62 / 79 / 196 / 696 / 862, 37 / 62 / 523, 37 / 196, 37 / 528 / 790, 39, 39 / 70 / 109 / 830 / 842, 39 / 70 / 725, 39 / 267, 39 / 267 / 489 / 522 / 612 / 830 / 842, 39 / 267 / 489 / 830 / 944, 40, 70 / 267 / 725 / 944 The substitution includes at least one substitution in a position or set of positions selected from 70 / 267 / 930 / 944, 70 / 489 / 930, 107, 109, 109 / 522 / 830 / 944, 217, 267 / 489 / 500 / 725 / 830 / 930, 267 / 522 / 725, 352, 385, 500 / 930 / 944, 673, 734, 774, 778, 875, 930, 932, and 934, where these positions are numbered with reference to Sequence ID No. 18.In some embodiments, acid alpha-glucosidase is 22R, 24E, 24R, 24W, 27G, 27G / 165I, 27K, 27R, 27W, 30D, 33G, 34D, 34M, 34T, 37F / 62E, 37F / 62E / 79S / 196T / 696S / 862Q, 37F / 62E / 523N , 37F / 196T, 37F / 528S / 790V, 39D, 39Q, 39Q / 70A / 109P / 830K / 842S, 39Q / 70A / 725E , 39Q / 267K, 39Q / 267K / 489D / 522V / 612D / 830K / 842S, 39Q / 267K / 489D / 830K / 944S, The configuration includes at least one substitution in a position or set of positions selected from 40W, 70A / 267K / 725E / 944S, 70A / 267K / 930P / 944S, 70A / 489D / 930P, 107G, 109D, 109P / 522V / 830K / 944S, 217D, 267K / 489D / 500A / 725E / 830K / 930P, 267K / 522V / 725E, 352K, 352V, 385G, 500A / 930P / 944S, 673N, 734K, 774G, 778Q, 875D, 930P, 932A, and 934F, where these positions are numbered with reference to Sequence ID No. 18.In some embodiments, the acid alpha-glucosidase is I22R, L24E, L24R, L24W, F27G, F27G / M165I, F27K, F27R, F27W, V30D, E33G, L34D, L34M, L34T, S37F / A62E, S37F / A62E / N79S / A196T / A696S / R862Q, S37F / A62E / D523 N, S37F / A196T, S37F / N528S / I790V, P39D, P39Q, P39Q / V70A / L109P / Q830K / G842S, P39Q / V70A / K 725E, P39Q / R267K, P39Q / R267K / A489D / E522V / S612D / Q830K / G842S, P39Q / R267K / A489D / Q830K / C944S, V40W, V70A / R267K / K725E / C944S, V70A / R267K / C930P / C944S, V70A / A489D / C930P, Q107G , L109D, L109P / E522V / Q830K / C944S, Q217D, R267K / A489D / D500A / K725E / Q830K / C930P, R267K / E The substitution includes at least one substitution in a position or set of positions selected from 522V / K725E, Y352K, Y352V, R385G, D500A / C930P / C944S, P673N, H734K, A774G, L778Q, N875D, C930P, S932A, and L934F, where these positions are numbered with reference to Sequence ID No. 18.

[0014] The present invention also provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to Sequence ID No. 20, the position of which is numbered with reference to Sequence ID No. 20. In some embodiments, acid alpha-glucosidase is 22 / 24, 22 / 24 / 27 / 50 / 107 / 109 / 489 / 612 / 725, 22 / 24 / 27 / 489, 22 / 24 / 27 / 489 / 612 / 774, 22 / 24 / 27 / 612 / 944, 22 / 24 / 50 / 109 / 267 / 489 / 774 / 944, 22 / 24 / 50 / 267 / 612 / 922 / 944, 22 / 24 / 107 / 267 / 489 / 92 2, 22 / 24 / 489, 22 / 24 / 612 / 725 / 944, 22 / 50 / 107 / 267 / 489 / 612 / 944, 22 / 50 / 109 / 267 / 489, 22 / 267 / 489 / 612, 24, 24 / 27 / 50 / 107 / 267 / 774 / 944, 24 / 27 / 89 / 500 / 842, 24 / 27 / 107 / 267 / 612 / 944, 24 / 27 / 267 / 944, 24 / 27 / 500 / 842, 24 / 27 / 500 / 842 / 932, 24 / 27 / 944, 24 / 39 / 49 / 89 / 97 / 842 / 932, 24 / 39 / 68 / 89 / 107 / 500 / 842, 24 / 39 / 89 / 97 / 842 / 932, 24 / 39 / 842 / 932, 24 / 50 / 489 / 944, 24 / 50 / 612, 24 / 70 / 107 / 109 / 489 / 612 / 725, 24 / 70 / 267 / 774, 24 / 89 / 500, 24 / 107 / 109 / 267 / 489 / 612 / 725 / 774, 24 / 109 / 612, 24 / 109 / 944, 24 / 267 / 725 / 944, 24 / 489 / 944, 24 / 725, 24 / 842 / 932, 24 / 944, 27 / 39 / 49 / 97 / 500 / 842, 27 / 49 / 68 / 500 / 842, 34 / 39 / 500 / 932, 39 / 89 / 97 / 500, 42, 48, 50 / 109 / 489 / 612, 50 / 489 / 774,50 / 612 / 944, 57, 62, 68, 68 / 89 / 97 / 932, 71, 88, 89 / 97 / 107, 89 / 97 / 500, 89 / 842, 107 / 109, 107 / 500 / 842, 108, 109 / 612 / 774 / 944, 112, 123, 124, 148, 188, 193, 197, 204, 253, 264, 305, 312, 333, 381, 402, 402 / 781, 489, 489 / 944 , includes at least one substitution in a position or set of positions selected from 500 / 842, 500 / 932, 523, 527, 612, 612 / 725 / 944, 612 / 922, 614, 727, 742, 748, 820, 823, 832, 842 / 932, 858, 862, 911, 913, 914, 916, 923, 937, and 940, where these positions are numbered with reference to Sequence ID No. 20. In some embodiments, the acid alpha-glucosidase is 22R / 24W, 22R / 24W / 27A / 50V / 107G / 109D / 489A / 612S / 725E, 22R / 24W / 27A / 489A, 22R / 24W / 27A / 489A / 612S / 774S, 22R / 24W / 27A / 612S / 944R, 22R / 24W / 50V / 109D / 267R / 489A / 774S / 944R, 22R / 24W / 50V / 267R / 612S / 922E / 944R, 22R / 24W / 107G / 267R / 489A / 922 E, 22R / 24W / 489A, 22R / 24W / 612S / 725E / 944R, 22R / 50V / 107G / 267R / 489A / 612S / 944S, 22R / 50V / 109D / 267R / 489A, 22R / 267R / 489A / 612S, 24R, 24R / 27G / 89R / 500A / 842G, 24R / 27G / 500A / 842G, 24R / 27G / 500A / 842G / 932A, 24R / 39D / 68S / 89R / 107G / 500A / 842G, 24R / 39H / 49G / 89R / 97G / 842G / 932A, 24R / 39H / 89R / 97D / 842G / 932A, 24R / 39H / 842G / 932A, 24R / 89R / 500A, 24R / 842G / 932A, 24W , 24W / 27A / 50V / 107G / 267R / 774S / 944S, 24W / 27A / 107G / 267R / 612S / 944S, 24W / 27A / 267R / 944R,24W / 27A / 944R, 24W / 50V / 489A / 944S, 24W / 50V / 612S, 24W / 70A / 107G / 109D / 489A / 612S / 725E, 24W / 70A / 267R / 774S, 24W / 107G / 109D / 267R / 489A / 612S / 725E / 774S, 24W / 109D / 612S, 24W / 109D / 944S, 24W / 267R / 725E / 944S, 24W / 489A / 944R, 24W / 725E, 24W / 944S, 27G / 39H / 49G / 97G / 500A / 842G, 27G / 49G / 68S / 500A / 842G, 34T / 39D / 500A / 932A, 39D / 89R / 97G / 500A, 42G, 48Q, 48V , 48W, 50V / 109D / 489A / 612S, 50V / 489A / 774S, 50V / 612S / 944S, 57F, 57L, 57 M, 62F, 62L, 62W, 68N / 89R / 97G / 932A, 68S, 71G, 71L, 71V, 71W, 71Y, 88L, 88R , 89R / 97G / 107G, 89R / 97G / 500A, 89R / 842G, 107G / 109D, 107G / 500A / 842G, 1 08R, 109D / 612S / 774S / 944S, 112H, 123L, 123V, 124G, 124M, 124V, 148K, 14 8R, 188R, 188W, 193E, 193P, 197G, 204A, 253M, 264M, 305F, 312A, 333L, 381R , 381V, 381W, 402N, 402V / 781Q, 489A, 489A / 944R, 500A / 842G, 500A / 932A, 523E, 527R, 527V, 612S, 612S / 725E / 944S, 612S / 922E, 614Q, 614R, 614W, 72 The system includes at least one substitution in a position or set of positions selected from 7W, 742V, 748V, 820A, 820V, 823F, 823V, 832A, 832R, 842G / 932A, 858C, 858W, 862I, 862M, 862Q, 862Y, 911G, 911R, 913G, 913R, 913W, 914G, 914I, 914K, 914Q, 914R, 914S, 914T, 916G, 916H, 916R, 923L, 923V, 923W, 937K, and 940Q, where these positions are numbered with reference to Sequence ID No. 20. In some embodiments,Acid alpha-glucosidases include I22R / L24W, I22R / L24W / F27A / Q50V / Q107G / L109D / D489A / D612S / K725E, I22R / L24W / F27A / D489A, I22R / L24W / F27A / D489A / D612S / A774S, I22R / L24W / F27A / D612S / C944R, I22R / L24W / Q50V / L109D / K267R / D489A / A774S / C944R, I22R / L24W / Q50V / K267R / D612S / P922E / C944R, and I22R / L 24W / Q107G / K267R / D489A / P922E, I22R / L24W / D489A, I22R / L24W / D612S / K7 25E / C944R, I22R / Q50V / Q107G / K267R / D489A / D612S / C944S, I22R / Q50V / L1 09D / K267R / D489A, I22R / K267R / D489A / D612S, L24R, L24R / F27G / A89R / D50 0A / S842G, L24R / F27G / D500A / S842G, L24R / F27G / D500A / S842G / S932A, L24 R / Q39D / R68S / A89R / Q107G / D500A / S842G, L24R / Q39H / Q49G / A89R / A97G / S8 42G / S932A, L24R / Q39H / A89R / A97D / S842G / S932A, L24R / Q39H / S842G / S932 A, L24R / A89R / D500A, L24R / S842G / S932A, L24W, L24W / F27A / Q50V / Q107G / K 267R / A774S / C944S, L24W / F27A / Q107G / K267R / D612S / C944S, L24W / F27A / K 267R / C944R, L24W / F27A / C944R, L24W / Q50V / D489A / C944S, L24W / Q50V / D61 2S, L24W / V70A / Q107G / L109D / D489A / D612S / K725E, L24W / V70A / K267R / A77 4S, L24W / Q107G / L109D / K267R / D489A / D612S / K725E / A774S, L24W / L109D / D 612S, L24W / L109D / C944S, L24W / K267R / K725E / C944S, L24W / D489A / C944R,L24W / K725E, L24W / C944S, F27G / Q39H / Q49G / A97G / D500A / S842G, F27G / Q49G / R68S / D500A / S842G, L34T / Q39D / D500A / S932A, Q39D / A89R / A97G / D500A, E42G, H48Q, H48V, H48W, Q50V / L109D / D489A / D612S, Q50V / D489A / A774S, Q50V / D612S / C944S, P57F, P57L, P57M, A62F, A62L, A62W, R68N / A89R / A97G / S932A, R68S, P71G, P71L, P71V, P71W, P71Y, K88L, K88R, A89 R / A97G / Q107G, A89R / A97G / D500A, A89R / S842G, Q107G / L109D, Q107G / D 500A / S842G, G108R, L109D / D612S / A774S / C944S, A112H, P123L, P123V, S124G, S124M, S124V, T148K, T148R, E188R, E188W, H193E, H193P, P197G , E204A, A253M, S264M, L305F, V312A, I333L, E381R, E381V, E381W, S402 N, S402V / P781Q, D489A, D489A / C944R, D500A / S842G, D500A / S932A, D52 3E, N527R, N527V, D612S, D612S / K725E / C944S, D612S / P922E, E614Q, E6 14R, E614W, S727W, L742V, L748V, G820A, G820V, L823F, L823V, P832A, P The substitution includes at least one substitution in a position or set of positions selected from 832R, S842G / S932A, E858C, E858W, R862I, R862M, R862Q, R862Y, N911G, N911R, V913G, V913R, V913W, P914G, P914I, P914K, P914Q, P914R, P914S, P914T, S916G, S916H, S916R, D923L, D923V, D923W, E937K, and L940Q, where these positions are numbered with reference to Sequence ID No. 20. In some additional embodiments, the acid alpha-glucosidase is 22 / 24,22 / 24 / 27 / 50 / 107 / 109 / 489 / 612 / 725、22 / 24 / 27 / 489、22 / 24 / 27 / 489 / 612 / 774、22 / 24 / 27 / 612 / 944、22 / 24 / 50 / 109 / 267 / 489 / 774 / 944、22 / 24 / 50 / 267 / 612 / 922 / 944、22 / 24 / 107 / 267 / 489 / 922、 ,22 / 24 / 489,22 / 24 / 612 / 725 / 944,22 / 50 / 107 / 267 / 489 / 612 / 944,22 / 50 / 109 / 267 / 489,22 / 267 / 489 / 612,24,24 / 27 / 50 / 107 / 267 / 774 / 944,24 / 27 / 89 / 500 / 842,24 / 27 / 107 / 267 / 612 / 944,24 / 27 / 267 / 944,24 / 27 / 500 / 842,24 / 27 / 500 / 842 / 932,24 / 27 / 944,24 / 39 / 49 / 89 / 97 / 842 / 932,24 / 39 / 68 / 89 / 107 / 500 / 842, 24 / 39 / 89 / 97 / 842 / 932, 24 / 39 / 842 / 932, 24 / 50 / 489 / 944, 24 / 50 / 612, 24 / 70 / 107 / 109 / 489 / 612 / 725, 24 / 70 / 267 / 774, 24 / 89 / 500, 24 / 107 / 109 / 267 / 489 / 612 / 725 / 774, 24 / 109 / 612, 24 / 109 / 944, 24 / 267 / 725 / 944, 24 / 489 / 944, 24 / 725, 24 / 842 / 932, 24 / 944, 27 / 3 9 / 49 / 97 / 500 / 842, 27 / 49 / 68 / 500 / 842, 34 / 39 / 500 / 932, 50 / 109 / 489 / 612, 50 / 612 / 944, 68 / 89 / 97 / 932, 89 / 97 / 107, 89 / 842, 106, 107 / 109, 107 / 500 / 842, 108, 109 / 612 / 774 / 944, 112, 148, 148 / 772, 188 / 377, 238, 240, 240 / 374, 243, 244, 246, 248, 249 / 777, 252, 253, 259, 260, 261, 262, 26 The substitution includes at least one substitution in a position or set of positions selected from 4, 279, 305, 309, 312, 319, 320, 329, 333, 387, 402, 421, 432, 500 / 842, 500 / 932, 556, 612, 612 / 725 / 944, 612 / 922, 727, 736, 737, 741, 742, 748, 815, 816, 818, 823, 832, 842 / 932, 911, 913, 914, 916, 923, 937, and 940, where these positions are numbered with reference to Sequence ID No. 20. In some embodiments, the acid alpha-glucosidase is 22R / 24W,22R / 24W / 27A / 50V / 107G / 109D / 489A / 612S / 725E、22R / 24W / 27A / 489A、22R / 24W / 27A / 489A / 612S / 774S、22R / 24W / 27A / 612S / 944R、22R / 24W / 50V / 109D / 267R / 489A / 774S / 944R、22R / 24W / 50V / 267R / 612S / 922E / 944R、22R / 24W / 107G / 267R / 489A / 922E、22R / 24W / 489A、22R / 24W / 612S / 725E / 944R、22R / 50V / 107G / 267R / 489A / 612S / 944S、22R / 50V / 109D / 267R / 489A、22R / 267R / 489A / 612S、24R、24R / 27G / 89R / 500A / 842G、24R / 27G / 500A / 842G、24R / 27G / 500A / 842G / 932A、24R / 39D / 68S / 89R / 107G / 500A / 842G、24R / 39H / 49G / 89R / 97G / 842G / 932A、24R / 39H / 89R / 97D / 842G / 932A、24R / 39H / 842G / 932A、24R / 89R / 500A、24R / 842G / 932A、24W、24W / 27A / 50V / 107G / 267R / 774S / 944S、24W / 27A / 107G / 267R / 612S / 944S、24W / 27A / 267R / 944R、24W / 27A / 944R、24W / 50V / 489A / 944S、24W / 50V / 612S、24W / 70A / 107G / 109D / 489A / 612S / 725E、24W / 70A / 267R / 774S、24W / 107G / 109D / 267R / 489A / 612S / 725E / 774S、24W / 109D / 612S、24W / 109D / 944S、24W / 267R / 725E / 944S、24W / 489A / 944R、24W / 725E、24W / 944S、27G / 39H / 49G / 97G / 500A / 842G、27G / 49G / 68S / 500A / 842G、34T / 39D / 500A / 932A、50V / 109D / 489A / 612S、50V / 612S / 944S、68N / 89R / 97G / 932A、89R / 97G / 107G、89R / 842G、106A、106G、106N、106T、107G / 109D、107G / 500A / 842G、108H、108N, 108R, 108S, 108V, 109D / 612S / 774S / 944S, 112H, 112P, 148E, 148G, 148H, 148K, 148R / 772I, 188Q / 377Q, 238Q , 240I, 240W / 374T, 240Y, 243E, 243G, 243R, 243V, 244I, 244V, 246A, 246G, 248A, 248R, 248V, 249V / 777N, 252V, 253 G, 253P, 259G, 259N, 259S, 260W, 261E, 262P, 264C, 279E, 305F, 305G, 305R, 305V, 305Y, 309C, 309G, 312A, 319F, 32 0M, 329F, 333L, 333V, 387L, 402G, 402N, 421P, 432C, 500A / 842G, 500A / 932A, 556H, 556R, 556S, 556Y, 612S, 612S / 72 5E / 944S, 612S / 922E, 727G, 727Q, 727T, 727W, 736M, 736V, 736W, 737M, 741C, 741D, 741E, 741G, 741T, 742V, 748I, 7 48T, 748V, 815A, 815M, 816V, 818T, 818V, 823A, 823F, 823G, 823R, 832E, 832G, 842G / 932A, 911G, 913A, 913E, 913G, The substitution includes at least one substitution in a position or set of positions selected from 913H, 913L, 913Q, 913R, 913W, 914E, 914G, 914H, 914K, 914Q, 914R, 914S, 914T, 916A, 916G, 916H, 916I, 916R, 916V, 923W, 937Q, 940G, 940Q, 940T, and 940W, where these positions are numbered with reference to sequence number 20. In some embodiments, the acid alpha-glucosidase is I22R / L24W, I22R / L24W / F27A / Q50V / Q107G / L109D / D489A / D612S / K725E, I22R / L24W / F27A / D489A, I22R / L24W / F27A / D489A / D612S / A774S, I22R / L24W / F27A / D612S / C944R, I22R / L24W / Q50V / L109D / K267R / D489A / A774S / C944R,I22R / L24W / Q50V / K267R / D612S / P922E / C944R、I22R / L24W / Q107G / K267R / D489A / P922E、I22R / L24W / D489A、I22R / L24W / D612S / K725E / C944R、I22R / Q50V / Q107G / K267R / D489A / D612S / C944S、I22R / Q50V / L109D / K267R / D489A、I22R / K267R / D489A / D612S、L24R、L24R / F27G / A89R / D500A / S842G、L24R / F27G / D500A / S842G、L24R / F27G / D500A / S842G / S932A、L24R / Q39D / R68S / A89R / Q107G / D500A / S842G、L24R / Q39H / Q49G / A89R / A97G / S842G / S932A、L24R / Q39H / A89R / A97D / S842G / S932A、L24R / Q39H / S842G / S932A、L24R / A89R / D500A、L24R / S842G / S932A、L24W、L24W / F27A / Q50V / Q107G / K267R / A774S / C944S、L24W / F27A / Q107G / K267R / D612S / C944S、L24W / F27A / K267R / C944R、L24W / F27A / C944R、L24W / Q50V / D489A / C944S、L24W / Q50V / D612S、L24W / V70A / Q107G / L109D / D489A / D612S / K725E、L24W / V70A / K267R / A774S、L24W / Q107G / L109D / K267R / D489A / D612S / K725E / A774S、L24W / L109D / D612S、L24W / L109D / C944S、L24W / K267R / K725E / C944S、L24W / D489A / C944R、L24W / K725E、L24W / C944S、F27G / Q39H / Q49G / A97G / D500A / S842G、F27G / Q49G / R68S / D500A / S842G、L34T / Q39D / D500A / S932A、Q50V / L109D / D489A / D612S、Q50V / D612S / C944S、R68N / A89R / A97G / S932A、A89R / A97G / Q107G、A89R / S842G、K106A、K106G、K106N、K106T, Q107G / L109D, Q107G / D500A / S842G, G108H, G108N, G108R, G108S, G108V, L109D / D612S / A774S / C944S, A112H, A112P, T148E, T148G, T1 48H, T148K, T148R / V772I, E188Q / R377Q, L238Q, L240I, L240W / A374T, L240Y, S243E, S243G, S243R, S243V, L244I, L244V, S246A, S246G, Y248A , Y248R, Y248V, I249V / S777N, L252V, A253G, A253P, L259G, L259N, L25 9S, M260W, L261E, S262P, S264C, P279E, L305F, L305G, L305R, L305V, L3 05Y, A309C, A309G, V312A, A319F, L320M, L329F, I333L, I333V, H387L, S402G, S402N, Q421P, M432C, D500A / S842G, D500A / S932A, F556H, F556R , F556S, F556Y, D612S, D612S / K725E / C944S, D612S / P922E, S727G, S72 7Q, S727T, S727W, L736M, L736V, L736W, L737M, A741C, A741D, A741E, A 741G, A741T, L742V, L748I, L748T, L748V, I815A, I815M, I816V, L818T , L818V, L823A, L823F, L823G, L823R, P832E, P832G, S842G / S932A, N911 The system includes at least one substitution in a position or set of positions selected from G, V913A, V913E, V913G, V913H, V913L, V913Q, V913R, V913W, P914E, P914G, P914H, P914K, P914Q, P914R, P914S, P914T, S916A, S916G, S916H, S916I, S916R, S916V, D923W, E937Q, L940G, L940Q, L940T, and L940W, where these positions are numbered with reference to sequence number 20.

[0015] The present invention further provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to Sequence ID No. 946, the position of which is numbered with reference to Sequence ID No. 946. In some embodiments, acid alpha-glucosidase is 19 / 124 / 149 / 381 / 727, 24 / 39 / 489 / 862, 24 / 57 / 62 / 89 / 489 / 823 / 862, 24 / 57 / 823 / 862, 24 / 62 / 89 / 188 / 823 / 842 / 862, 24 / 89 / 489, 24 / 89 / 489 / 727 / 862, 24 / 489 / 500 / 842, 39 / 57 / 62 / 188 / 500 / 842, 39 / 57 / 500 / 862, 57, 57 / 62 / 120 / 527 / 913 / 916, 57 / 62 / 305 / 437 / 500 / 614 / 727 / 916, 57 / 62 / 305 / 437 / 500 / 727 / 913 / 916, 57 / 62 / 305 / 437 / 614 / 683 / 913 / 916 / 932, 57 / 62 / 305 / 489 / 907 / 913 / 916, 57 / 62 / 305 / 489 / 913 / 916, 57 / 62 / 305 / 5 00 / 913 / 916, 57 / 62 / 305 / 913, 57 / 62 / 305 / 916, 57 / 62 / 437 / 500 / 761 / 914 / 916, 57 / 62 / 437 / 527 / 727, 57 / 62 / 437 / 913 / 916, 57 / 62 / 913 / 916 / 932, 57 / 62 / 916, 57 / 188 / 489 / 823 / 862, 57 / 305, 57 / 305 / 437 / 916, 57 / 437 / 500 / 527 / 727 / 916, 57 / 437 / 500 / 614 / 727 / 914, 57 / 437 / 913 / 914, 57 / 489 / 527 / 914 / 916, 57 / 614 / 916 / 932, 62 / 89, 62 / 89 / 124 / 148 / 381 / 858, 62 / 89 / 124 / 381 / 858, 62 / 89 / 148 / 381 / 614 / 858, 62 / 89 / 148 / 923, 62 / 89 / 149 / 381, 62 / 89 / 149 / 381 / 832,62 / 89 / 188 / 489 / 500 / 727 / 823, 62 / 89 / 381, 62 / 89 / 381 / 858, 62 / 89 / 381 / 923, 62 / 89 / 858, 62 / 96 / 614, 62 / 124 / 148 / 149 / 381 / 614, 62 / 124 / 149 / 381 / 832 / 858 / 937, 62 / 124 / 188 / 823 / 842 / 862, 62 / 124 / 381 / 832, 62 / 148 / 149 / 381 / 858 / 937, 62 / 148 / 381 / 61 4 / 937, 62 / 148 / 381 / 727, 62 / 148 / 381 / 858, 62 / 149 / 381 / 614 / 937, 62 / 149 / 381 / 858 / 937, 62 / 149 / 727, 62 / 305 / 437 / 500 / 727 / 913, 62 / 305 / 727, 62 / 381, 62 / 437 / 489 / 527 / 727 / 913 / 932, 62 / 437 / 489 / 614 / 727 / 913, 62 / 437 / 527 / 727, 62 / 437 / 527 / 916 / 932 , 62 / 437 / 913 / 916, 62 / 489 / 500 / 932, 62 / 489 / 527 / 916 / 932, 62 / 489 / 614 / 916, 62 / 500, 62 / 527, 62 / 527 / 727 / 916, 62 / 614, 62 / 727, 62 / 916, 89 / 148 / 149, 89 / 148 / 149 / 381, 89 / 381, 124 / 148 / 381 / 727 / 858 / 937, 124 / 381 / 614, 124 / 500 / 842 / 862, 124 / 832 / The position or set of positions selected from 937, 148 / 832 / 858 / 937, 381, 381 / 614 / 832, 381 / 858 / 937, 437, 437 / 489 / 914 / 916, 437 / 727 / 914, 437 / 914 / 916, 489 / 500, 489 / 614 / 916, 500 / 727 / 913 / 916, 500 / 914 / 916, and 923 includes at least one substitution, where these positions are numbered with reference to sequence number 946. In some embodiments, acid alpha-glucosidase is 19T / 124V / 149R / 381V / 727W, 24R / 39H / 489A / 862Q, 24R / 57L / 62W / 89R / 489A / 823F / 862Q, 24R / 57L / 823F / 862Q, 24R / 62W / 89R / 188W / 823V / 842G / 862Q, 24R / 89R / 489A,24R / 89R / 489A / 727W / 862Q、24R / 489A / 500A / 842G、39H / 57L / 62W / 188W / 500 A / 842G、39H / 57L / 500A / 862Q、57F / 62L / 305F / 437G / 500A / 614Q / 727W / 916R 57F / 62L / 305F / 437G / 614Q / 683S / 913R / 916R / 932A 57F / 62L / 305F / 500A / 913R / 916G 57F / 62L / 305F / 913R 57F / 62L / 437G / 500A / 761F / 914K / 916R 5 7F / 62L / 437G / 527R / 727W, 57F / 62L / 913R / 916R / 932A, 57F / 62L / 916G, 57F / 62W / 120I / 527R / 913R / 916R, 57F / 62W / 305F / 437G / 500A / 727W / 913R / 916R 57F / 62W / 305F / 489A / 907K / 913R / 916G、57F / 62W / 305F / 489A / 913R / 916G、57F / 62W / 305F / 916R、57F / 62W / 437G / 913R / 916G、57F / 305F、57F / 305F / 437G / 916G, 57F / 437G / 500A / 527R / 727W / 916R, 57F / 437G / 500A / 614Q / 727W / 914R, 57F / 437G / 913R / 914R, 57F / 489A / 527R / 914R / 916G, 57F / 614Q / 916G / 932A 、57L、57L / 188W / 489A / 823F / 862Q、62F / 89R、62F / 89R / 124V / 148R / 381W / 85 8W、62F / 89R / 148R / 381V / 614R / 858W、62F / 89R / 148R / 923W、62F / 89R / 149R / 381W / 832R、62F / 89R / 381V / 923W、62F / 89R / 858C、62F / 96K / 614R、62F / 124V / 381W / 832R、62F / 149R / 381V / 858W / 937K、62F / 149R / 727W、62F / 381V、62F / 614R, 62L / 305F / 437G / 500A / 727W / 913R, 62L / 305F / 727W, 62L / 437G / 489A / 527R / 727W / 913R / 932A, 62L / 437G / 527R / 727W, 62L / 437G / 527R / 916G / 932A62L / 437G / 913R / 916R, 62L / 489A / 500A / 932A, 62L / 489A / 614Q / 916R, 62L / 527R, 62L / 527R / 727W / 916G, 62W / 89R / 124V / 381W / 858 C, 62W / 89R / 149R / 381W, 62W / 89R / 188W / 489A / 500A / 727W / 823F, 62W / 89R / 381V, 62W / 89R / 381W / 858C, 62W / 124V / 148R / 149R / 381 W / 614R, 62W / 124V / 149R / 381V / 832R / 858C / 937K, 62W / 124V / 188W / 823F / 842G / 862Q, 62W / 148R / 149R / 381V / 858C / 937K, 62W / 148 R / 381W / 614R / 937K, 62W / 148R / 381W / 727W, 62W / 148R / 381W / 858C, 62W / 149R / 381W / 614R / 937K, 62W / 381V, 62W / 437G / 489A / 614Q / 727W / 913R,62W / 489A / 527R / 916R / 932A,62W / 500A,62W / 727W,62W / 916G,89R / 148R / 149R,89R / 148R / 149R / 381W,89R / 381W,12 4V / 148R / 381W / 727W / 858W / 937K, 124V / 381W / 614R, 124V / 500A / 842G / 862Q, 124V / 832R / 937K, 148R / 832R / 858W / 937K, 381V, 381 The substitution includes at least one substitution in a position or set of positions selected from V / 614R / 832R, 381W / 858C / 937K, 437G, 437G / 489A / 914R / 916R, 437G / 727W / 914K, 437G / 914R / 916G, 489A / 500A, 489A / 614Q / 916G, 500A / 727W / 913R / 916R, 500A / 914R / 916G, and 923W, where these positions are numbered with reference to sequence number 946. In some embodiments, the acidic alpha-glucosidase is S19T / S124V / P149R / E381V / S727W, W24R / Q39H / D489A / R862Q, W24R / P57L / A62W / A89R / D489A / L823F / R862Q, W24R / P57L / L823F / R862Q,W24R / A62W / A89R / E188W / L823V / S842G / R862Q、W24R / A89R / D489A、W24R / A89R / D489A / S727W / R862Q、W24R / D489A / D500A / S842G、Q39H / P57L / A62W / E188W / D500A / S842G、Q39H / P57L / D500A / R862Q、P57F / A62L / L305F / A437G / D500A / E614Q / S727W / S916R、P57F / A62L / L305F / A437G / E614Q / A683S / V913R / S916R / S932A、P57F / A62L / L305F / D500A / V913R / S916G、P57F / A62L / L305F / V913R、P57F / A62L / A437G / D500A / L761F / P914K / S916R、P57F / A62L / A437G / N527R / S727W、P57F / A62L / V913R / S916R / S932A、P57F / A62L / S916G、P57F / A62W / F120I / N527R / V913R / S916R、P57F / A62W / L305F / A437G / D500A / S727W / V913R / S916R、P57F / A62W / L305F / D489A / Q907K / V913R / S916G、P57F / A62W / L305F / D489A / V913R / S916G、P57F / A62W / L305F / S916R、P57F / A62W / A437G / V913R / S916G、P57F / L305F、P57F / L305F / A437G / S916G、P57F / A437G / D500A / N527R / S727W / S916R、P57F / A437G / D500A / E614Q / S727W / P914R、P57F / A437G / V913R / P914R、P57F / D489A / N527R / P914R / S916G、P57F / E614Q / S916G / S932A、P57L、P57L / E188W / D489A / L823F / R862Q、A62F / A89R、A62F / A89R / S124V / T148R / E381W / E858W、A62F / A89R / T148R / E381V / E614R / E858W、A62F / A89R / T148R / D923W、A62F / A89R / P149R / E381W / P832R、A62F / A89R / E381V / D923W、A62F / A89R / E858C、A62F / E96K / E614R、A62F / S124V / E381W / P832R、A62F / P149R / E381V / E858W / E937K、A62F / P149R / S727W、A62F / E381V、A62F / E614R、A62L / L305F / A437G / D500A / S727、 W / V913R、A62L / L305F / S727W、A62L / A437G / D489A / N527R / S727W / V913R / S9 32A、A62L / A437G / N527R / S727W、A62L / A437G / N527R / S916G / S932A、A62L / A 437G / V913R / S916R、A62L / D489A / D500A / S932A、A62L / D489A / E614Q / S916R 、A62L / N527R、A62L / N527R / S727W / S916G、A62W / A89R / S124V / E381W / E858C、 A62W / A89R / P149R / E381W、A62W / A89R / E188W / D489A / D500A / S727W / L823F、 A62W / A89R / E381V、A62W / A89R / E381W / E858C、A62W / S124V / T148R / P149R / E3 81W / E614R、A62W / S124V / P149R / E381V / P832R / E858C / E937K、A62W / S124V / E188W / L823F / S842G / R862Q、A62W / T148R / P149R / E381V / E858C / E937K、A62W / T148R / E381W / E614R / E937K、A62W / T148R / E381W / S727W、A62W / T148R / E38 1W / E858C、A62W / P149R / E381W / E614R / E937K、A62W / E381V、A62W / A437G / D4 89A / E614Q / S727W / V913R、A62W / D489A / N527R / S916R / S932A、A62W / D500A、 A62W / S727W、A62W / S916G、A89R / T148R / P149R、A89R / T148R / P149R / E381W、A 89R / E381W、S124V / T148R / E381W / S727W / E858W / E937K、S124V / E381W / E614 R、S124V / D500A / S842G / R862Q、S124V / P832R / E937K、T148R / P832R / E858W / E 937K、E381V、E381V / E614R / P832R、E381W / E858C / E937K、A437G、A437G / D48 9A / P914R / S916R、A437G / S727W / P914K、A437G / P914R / S916G、D489A / D500A、The substitution includes at least one substitution in a position or set of positions selected from D489A / E614Q / S916G, D500A / S727W / V913R / S916R, D500A / P914R / S916G, and D923W, where these positions are numbered with reference to sequence number 946. In some embodiments, acid alpha-glucosidase is 57 / 62 / 120 / 527 / 913 / 916, 57 / 62 / 305 / 437 / 500 / 614 / 727 / 916, 57 / 62 / 305 / 437 / 500 / 727 / 913 / 916, 57 / 62 / 305 / 437 / 614 / 683 / 913 / 916 / 932, 57 / 62 / 305 / 489 / 907 / 913 / 916, 57 / 62 / 305 / 489 / 913 / 916, 57 / 62 / 305 / 500 / 913 / 916, 57 / 62 / 305 / 913, 57 / 62 / 305 / 916, 57 / 62 / 437 / 500 / 761 / 914 / 916, 57 / 62 / 437 / 527 / 727, 57 / 62 / 437 / 913 / 916, 57 / 62 / 913 / 916 / 932, 57 / 62 / 916, 57 / 188 / 489 / 823 / 862, 57 / 305, 57 / 305 / 437 / 916, 57 / 437 / 500 / 527 / 727 / 916, 57 / 437 / 500 / 614 / 727 / 914, 57 / 437 / 913 / 9 14, 57 / 489 / 527 / 914 / 916, 57 / 614 / 916 / 932, 62 / 89 / 188 / 489 / 500 / 727 / 823, 62 / 124 / 188 / 823 / 842 / 862, 62 / 305 / 437 / 500 / 727 / 913, 62 / 305 / 727, 62 / 437 / 489 / 527 / 727 / 913 / 932, 62 / 437 / 489 / 614 / 727 / 913, 62 / 437 / 527 / 727, 62 / 437 / 527 / 916 / 932, 62 / 437 / 913 The system includes at least one substitution for a position or set of positions selected from / 916, 62 / 489 / 500 / 932, 62 / 489 / 527 / 916 / 932, 62 / 527 / 727 / 916, 62 / 727, 62 / 916, 124 / 500 / 842 / 862, 437, 437 / 489 / 914 / 916, 437 / 727 / 914, 437 / 914 / 916, 489 / 614 / 916, 500 / 727 / 913 / 916, 500 / 914 / 916, and 923, where these positions are:The numbers are numbered with reference to Sequence ID No. 946. In some embodiments, the acid alpha-glucosidases are 57F / 62L / 305F / 437G / 500A / 614Q / 727W / 916R, 57F / 62L / 305F / 437G / 614Q / 683S / 913R / 916R / 932A, 57F / 62L / 305F / 500A / 913R / 916G, 57F / 62L / 305F / 913R, 57F / 62L / 437G / 500A / 761F / 914K / 916R, 57F / 62L / 437G / 527R / 727W, 57F / 62L / 913R / 916R / 932A, 57F / 62L / 91 6G, 57F / 62W / 120I / 527R / 913R / 916R, 57F / 62W / 305F / 437G / 500A / 727W / 9 13R / 916R, 57F / 62W / 305F / 489A / 907K / 913R / 916G, 57F / 62W / 305F / 489A / 9 13R / 916G, 57F / 62W / 305F / 916R, 57F / 62W / 437G / 913R / 916G, 57F / 305F, 57 F / 305F / 437G / 916G, 57F / 437G / 500A / 527R / 727W / 916R, 57F / 437G / 500A / 6 14Q / 727W / 914R, 57F / 437G / 913R / 914R, 57F / 489A / 527R / 914R / 916G, 57F / 614Q / 916G / 932A, 57L / 188W / 489A / 823F / 862Q, 62L / 305F / 437G / 500A / 72 7W / 913R, 62L / 305F / 727W, 62L / 437G / 489A / 527R / 727W / 913R / 932A, 62L / 437G / 527R / 727W, 62L / 437G / 527R / 916G / 932A, 62L / 437G / 913R / 916R, 62L / 489A / 500A / 932A, 62L / 527R / 727W / 916G, 62W / 89R / 188W / 489A / 500A / 72 7W / 823F, 62W / 124V / 188W / 823F / 842G / 862Q, 62W / 437G / 489A / 614Q / 727W / 913R, 62W / 489A / 527R / 916R / 932A, 62W / 727W, 62W / 916G, 124V / 500A / 842G / 862Q, 437G, 437G / 489A / 914R / 916R, 437G / 727W / 914K, 437G / 914R / 916G,The substitution includes at least one substitution in a position or set of positions selected from 489A / 614Q / 916G, 500A / 727W / 913R / 916R, 500A / 914R / 916G, and 923W, where these positions are numbered with reference to sequence number 946. In some embodiments, the acidic alpha-glucosidase is P57F / A62L / L305F / A437G / D500A / E614Q / S727W / S916R, P57F / A62L / L305F / A437G / E614Q / A683S / V913R / S916R / S932A, P57F / A62L / L305F / D500A / V913R / S916G, P57F / A62L / L305F / V913R, P57F / A62L / A437G / D500A / L761F / P914K / S916 R, P57F / A62L / A437G / N527R / S727W, P57F / A62L / V913R / S916R / S932A, P57F / A62L / S916G, P57F / A62W / F120I / N527R / V913R / S916R, P 57F / A62W / L305F / A437G / D500A / S727W / V913R / S916R, P57F / A62W / L305F / D489A / Q907K / V913R / S916G, P57F / A62W / L305F / D489A / V9 13R / S916G, P57F / A62W / L305F / S916R, P57F / A62W / A437G / V913R / S916G, P57F / L305F, P57F / L305F / A437G / S916G, P57F / A437G / D500 A / N527R / S727W / S916R, P57F / A437G / D500A / E614Q / S727W / P914R, P57F / A437G / V913R / P914R, P57F / D489A / N527R / P914R / S916G, P5 7F / E614Q / S916G / S932A, P57L / E188W / D489A / L823F / R862Q, A62L / L305F / A437G / D500A / S727W / V913R, A62L / L305F / S727W, A62L / A4 37G / D489A / N527R / S727W / V913R / S932A, A62L / A437G / N527R / S727W, A62L / A437G / N527R / S916G / S932A, A62L / A437G / V913R / S916R,A62L / D489A / D500A / S932A, A62L / N527R / S727W / S916G, A62W / A89R / E188W / D489A / D500A / S727W / L823F, A62W / S124V / E188W / L823 F / S842G / R862Q, A62W / A437G / D489A / E614Q / S727W / V913R, A62W / D489A / N527R / S916R / S932A, A62W / S727W, A62W / S916G, S124V / D The sequence includes at least one substitution in a position or set of positions selected from 500A / S842G / R862Q, A437G, A437G / D489A / P914R / S916R, A437G / S727W / P914K, A437G / P914R / S916G, D489A / E614Q / S916G, D500A / S727W / V913R / S916R, D500A / P914R / S916G, and D923W, where these positions are numbered with reference to sequence number 946.

[0016] The present invention provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to Sequence ID No. 1956, where the position is numbered with reference to Sequence ID No. 1956. In some embodiments, acid alpha-glucosidase is 3 / 569, 41 / 53, 44, 44 / 347, 56, 65, 78, 78 / 87 / 176 / 266 / 536 / 615, 78 / 87 / 266, 78 / 87 / 266 / 372 / 386 / 777, 78 / 87 / 266 / 372 / 536, 78 / 87 / 266 / 483 / 924, 78 / 87 / 483 / 777, 78 / 87 / 536, 78 / 266 / 483 / 53 6 / 615, 78 / 266 / 483 / 795, 78 / 266 / 763, 78 / 372 / 390, 78 / 390, 78 / 536 / 615, 87, 87 / 266, 87 / 266 / 372 / 483, 87 / 266 / 483, 87 / 266 / 924, 87 / 372 / 777, 87 / 536 / 777, 87 / 615, 87 / 795, 105, 136, 141, 145, 154 / 588, 156, 157, 199, 202, 222, 225, 227, 229, 266, 266 / 372 / 536 / 615 / 763 / 777, 266 / 372 / 924, 266 / 536 / 615 / 795, 344, 348, 390 / 615, 412, 423, 425 / 678 / 894, 430, 446, 484, 488, 496, 499 / 711, 503, 530, 543, 569, 572, 573, 574, 577, 578, 579, 580, 581, 583, 585, 588, 589 / 663, 615, 628, 629, 631, 633, 656, 663, 669, 670, 671, 678, 679, 687, 690, 691, 692, 693, 705, 706, 708, 709, 710, 711, 726, 768, 773, 777, 779, 795, 797, 816, 826, 834, 857, 859, 868, 869, 871, 873, 877, 878,The positions or sets of positions selected from 909 include at least one substitution, and these positions are numbered with reference to sequence number 1956. In some embodiments, the acidic alpha-glucosidase is 3L / 569F, 41I / 53M, 44E, 44G, 44L, 44P / 347I, 44R, 56A, 56L, 56R, 65L, 65V, 78E, 78E / 87E / 176T / 266N / 536T / 615D, 78E / 87E / 266N, 78E / 87E / 266N / 372T / 386Y / 777G, 78E / 87E / 266N / 372T / 536T, 78E / 87E / 266N / 483S / 924N, 78E / 87E / 483S / 777G, 78E / 87E / 536T, 78E / 266N / 483S / 536T / 615D, 78E / 266N / 483S / 795E, 78E / 266N / 763L, 78E / 372T / 390Q, 78E / 390Q, 78E / 536T / 615D, 87E, 87E / 266N, 87E / 266N / 372T / 483S, 87E / 266N / 483S, 87E / 266N / 924N, 87E / 372T / 777G, 87E / 536T / 777G, 87E / 615D, 87E / 795E, 105T, 136G, 141S, 141W, 145I, 1 45R, 154R / 588L, 156L, 157S, 199V, 202K, 202L, 202N, 202R, 202T, 222C , 222P, 225D, 227A, 229C, 266N, 266N / 372T / 536T / 615D / 763L / 777G, 26 6N / 372T / 924N, 266N / 536T / 615D / 795E, 344G, 344M, 348G, 390Q / 615D, 412Y, 423V, 425R / 678I / 894C, 430F, 446T, 484L, 488G, 488K, 488M, 496 G,499Y / 711F,503S,503T,530V,543C,543Q,543S,543V,569H,569I,569Q,569S,569T,569V,569Y,572G,572S,573C,573D,573H,573M,573Q , 574S, 577A, 577D, 577E, 577M, 577T, 577V, 578S, 579V, 580E, 580G, 58 0I, 580L, 580W, 580Y, 581F, 581G, 581H, 581L, 581S, 581T, 581V, 581Y,583C, 583G, 583K, 583L, 585F, 585L, 585M, 585Q, 585V, 588L, 588V, 589I / 663F, 615G, 628I, 628M, 628V , 629A, 629C, 629G, 629I, 631I, 631L, 631M, 633V, 656M, 656V, 663A, 663F, 669R, 670E, 670F, 670I, 670Q , 670R, 670S, 670T, 670V, 670W, 671A, 671G, 671M, 671T, 678H, 678L, 678T, 678Y, 679W, 687L, 690V, 691 F, 691V, 692C, 692F, 692G, 692I, 692L, 692R, 692S, 692V, 692Y, 693F, 693I, 693Y, 705M, 706F, 706M, 706 V, 708C, 709S, 710K, 710L, 710M, 710N, 710S, 711C, 711F, 711G, 711H, 711L, 711R, 711W, 726E, 768S, 77 3V, 777G, 777I, 777R, 779E, 779H, 779R, 795E, 797L, 797M, 816L, 826G, 834H, 857T, 859T, 859Y, 868I, 86 The substitution includes at least one substitution in a position or set of positions selected from 8L, 869L, 869S, 871E, 871K, 871R, 873A, 873F, 873Y, 877L, 877V, 878A, 878F, 878G, 878K, 878L, 878Q, 878R, 878S, 878W, and 909F, where these positions are numbered with reference to Sequence ID No. 1956. In some embodiments, the acidic alpha-glucosidases are W3L / L569F, L41I / S53M, T44E, T44G, T44L, T44P / L347I, T44R, G56A, G56L, G56R, G65L, G65V, P78E, P78E / D87E / K176T / T266N / V536T / Q615D, P78E / D87E / T266N, P78E / D87E / T266N / S372T / A386Y / S777G, P78E / D87E / T266N / S372T / V536T, P78E / D87E / T266N / T483S / T924N, P78E / D87E / T483S / S777G, P78E / D87E / V536T, P78E / T266N / T483S / V536T / Q615D,P78E / T266N / T483S / Q795E, P78E / T266N / T763L, P78E / S372T / L390Q, P78E / L390Q, P78E / V536T / Q615D, D87E, D87E / T266N, D87E / T266N / S372T / T483S, D 87E / T266N / T483S, D87E / T266N / T924N, D87E / S372T / S777G, D87E / V536T / S777G, D87E / Q615D, D87E / Q795E, A105T, S136G, T141S, T141W, T145I, T145R K154R / F588L, I156L, L157S, P199V, S202K, S202L, S202N, S202R, S202T, V222C, V222P, N225D, T227A, A229C, T266N, T266N / S372T / V536T / Q615D / T763L / S777G、T266N / S372T / T924N、T266N / V536T / Q615D / Q795E、Q344G、Q344M、D 348G、L390Q / Q615D、G412Y、L423V、Q425R / S678I / G894C、Y430F、S446T、N484 L、L488G、L488K、L488M、A496G、H499Y / A711F、P503S、P503T、L530V、T543C、 T543Q、T543S、T543V、L569H、L569I、L569Q、L569S、L569T、L569V、L569Y、A57 2G, A572S, I573C, I573D, I573H, I573M, I573Q, A574S, R577A, R577D, R577E, R577M, R577T, R577V, A578S, L579V, V580E, V580G, V580I, V580L, V580W, V5 80Y、K581F、K581G、K581H、K581L、K581S、K581T、K581V、K581Y、R583C、R583 G、R583K、R583L、T585F、T585L、T585M、T585Q、T585V、F588L、F588V、V589I / M 663F、Q615G、L628I、L628M、L628V、L629A、L629C、L629G、L629I、V631I、V63 1L、V631M、L633V、L656M、L656V、M663A、M663F、L669R、L670E、L670F、L670I、L670Q, L670R, L670S, L670T, L670V, L670W, S671A, S671G, S671M, S671T, S678H, S678L, S6 78T, S678Y, F679W, M687L, A690V, L691F, L691V, T692C, T692F, T692G, T692I, T692L, T692R , T692S, T692V, T692Y, L693F, L693I, L693Y, F705M, H706F, H706M, H706V, A708C, H709S, V7 10K, V710L, V710M, V710N, V710S, A711C, A711F, A711G, A711H, A711L, A711R, A711W, D726E , Q768S, E773V, S777G, S777I, S777R, P779E, P779H, P779R, Q795E, V797L, V797M, I816L, T8 26G, A834H, L857T, V859T, V859Y, V868I, V868L, I869L, I869S, L871E, L871K, L871R, R873A The substitution includes at least one substitution in a position or set of positions selected from R873F, R873Y, I877L, I877V, V878A, V878F, V878G, V878K, V878L, V878Q, V878R, V878S, V878W, and L909F, where these positions are numbered with reference to Sequence ID No. 1956. In some embodiments, acid alpha-glucosidase is 44, 53, 56, 63, 65, 105, 125, 129, 136, 139, 141, 142, 145, 152, 156, 162, 176, 177, 185, 186, 187, 199, 199 / 775, 202, 265, 267, 337, 344, 348, 350, 354, 372, 373, 401, 412, 446, 469, 484, 488, 493, The substitution includes at least one substitution at a position selected from 496, 499, 503, 526, 543, 612, 615, 649, 677, 678, 679, 730, 752, 765, 768, 773, 777, 779, 788, 797, 822, 826, 834, 855, 856, 857, 859, 860, 924, 926, 931, and 936, where these positions are numbered with reference to Sequence ID No. 1956. In some embodiments, the acid alpha-glucosidase is 44A,44F、44V、44W、44Y、53I、56S、56W、63N、65A、65F、65R、65Y、105V、105W、125H、125W、129E、129I、129S、129T、129V、129W、136K、136R、136V、139E、141K、141R、142G、145A、145L、152L、152S、15、 2W, 156C, 156K, 156R, 156S, 162T, 176R, 177Q, 185L, 186H, 187I, 199A, 199G, 199I, 199R, 199T, 199V / 775I, 199W, 202A, 202 D, 202G, 202H, 202Q, 202Y, 265D, 265F, 265H, 267E, 267G, 267R, 337H, 344C, 348E, 348W, 350F, 350I, 354L, 354S, 372D, 373A, 373S, 401G, 401S, 412R, 412S, 412W, 446C, 446D, 446G, 446I, 446K, 469M, 469T, 469V, 484A, 484K, 484R, 488C, 488E, 488S, 49 3L, 496M, 496W, 499A, 499E, 499I, 499M, 499Q, 499V, 503C, 503H, 503N, 526L, 526V, 543G, 543H, 543K, 543L, 543R, 612G, 612L , 612R, 612T, 615M, 615S, 649M, 677T, 678Q, 678R, 678V, 678W, 679Y, 730K, 730L, 730R, 752F, 752G, 752L, 752N, 752S, 752W, 7 65W, 768I, 768K, 768V, 773P, 777M, 777W, 779I, 779M, 779S, 788A, 788H, 788I, 788L, 788N, 788Q, 788S, 788T, 788Y, 797E, 797 The positions include at least one substitution selected from F, 797I, 797R, 797W, 822R, 826I, 826M, 834G, 834S, 834V, 834W, 855G, 855L, 856A, 856G, 857A, 857E, 857R, 857S, 857V, 859A, 859G, 860S, 924A, 926M, 926T, 931L, 936N, 936R, and 936S, where these positions are numbered with reference to Sequence ID No. 1956. In some embodiments, the acidic alpha-glucosidases are T44A, T44F, T44V, T44W, T44Y, S53I, G56S, G56W, H63N, G65A, G65F, G65R, G65Y, A105V, A105W, Y125H, Y125W, K129E, K129I, K129S, K129T, K129V, K129W, S136K, S136R, S136V, G139E, T141K,T141R, A142G, T145A, T145L, F152L, F152S, F152W, I156C, I156K, I156R, I 156S, D162T, K176R, D177Q, V185L, P186H, L187I, P199A, P199G, P199I, P1 99R, P199T, P199V / L775I, P199W, S202A, S202D, S202G, S202H, S202Q, S20 2Y, W265D, W265F, W265H, K267E, K267G, K267R, P337H, Q344C, D348E, D348W , V350F, V350I, F354L, F354S, S372D, T373A, T373S, D401G, D401S, G412R, G412S, G412W, S446C, S446D, S446G, S446I, S446K, I469M, I469T, I469V, N4 84A, N484K, N484R, L488C, L488E, L488S, D493L, A496M, A496W, H499A, H49 9E, H499I, H499M, H499Q, H499V, P503C, P503H, P503N, P526L, P526V, T543G , T543H, T543K, T543L, T543R, S612G, S612L, S612R, S612T, Q615M, Q615S, L649M, Y677T, S678Q, S678R, S678V, S678W, F679Y, W730K, W730L, W730R, K 752F, K752G, K752L, K752N, K752S, K752W, Y765W, Q768I, Q768K, Q768V, E7 73P, S777M, S777W, P779I, P779M, P779S, P788A, P788H, P788I, P788L, P788 The substitution includes at least one substitution at a position selected from N, P788Q, P788S, P788T, P788Y, V797E, V797F, V797I, V797R, V797W, G822R, T826I, T826M, A834G, A834S, A834V, A834W, E855G, E855L, S856A, S856G, L857A, L857E, L857R, L857S, L857V, V859A, V859G, L860S, T924A, V926M, V926T, V931L, G936N, G936R, and G936S, where these positions include,The numbers are numbered with reference to Sequence ID No. 1956. In some embodiments, the acid alpha-glucosidases are 78 / 87 / 176 / 266 / 536 / 615, 78 / 87 / 266 / 372 / 386 / 777, 78 / 87 / 266 / 372 / 536, 78 / 266 / 763, 78 / 372 / 390, 87 / 266 / 372 / 483, 87 / 372 / 777, 105, 125, 129, 136, 139, 141, 142, 152, 154 / 588, 156, 222, 225, 227, 229, 266 / 372 / 536 / 615 / 763 / 777, 266 / 372 / 924, 267, 372, 401, 493 , 496, 499, 569, 572, 573, 574, 577, 579, 580, 581, 583, 585, 588, 589 / 663, 628, 629, 631, 663, 669, 670, 671, 691, 692, 693, 706, 708, 710, 711, 765, 768, 779, 797, 826, 834, 855, 856, 857, 869, 871, 873, 878, 909, 924, and 926, each containing at least one substitution in a position or set of positions numbered with reference to sequence number 1956. In some embodiments, the acidic alpha-glucosidase is 78E / 87E / 176T / 266N / 536T / 615D, 78E / 87E / 266N / 372T / 386Y / 777G, 78E / 87E / 266N / 372T / 536T, 78E / 266N / 763L, 78E / 372T / 390Q, 87E / 266N / 372T / 483S, 87E / 372T / 777G, 105T, 105W, 125W, 129E, 129S, 136G, 136K, 136V, 139E, 141S, 142G, 152S, 152W, 154R / 588L , 156C, 156L, 222C, 222P, 225D, 227A, 229C, 266N / 372T / 536T / 615D / 763L / 777G, 266N / 372T / 924N, 267E, 372D, 401G, 493L, 496G, 499E, 499I, 499M , 499Q, 569H, 569Q, 569S, 569T, 569V, 569Y, 572G, 572S, 573C, 573D, 573H , 573M, 573Q, 574S, 577A, 577D, 577E, 577T, 579V, 580E, 580G, 580W, 581G,581H, 581T, 583C, 583G, 585Q, 588L, 588V, 589I / 663F, 628V, 629A, 629C, 629G, 629I, 631I, 631L, 631M, 663A, 663F, 669R, 670E, 670F, 670I, 670Q, 670R, 670S, 670T, 670V, 670W, 671A, 671G, 671T, 691V, 692C, 692F, 692G, 692I, 692L, 692Y, 693F, 693I, 693Y, 706F, 708C, 710K, 710L, 710M, 710N, 710S, 711C, 711G, 711H, The position or set of positions selected from 711W, 765W, 768I, 779E, 797E, 797F, 797I, 797L, 797M, 797W, 826G, 834G, 834H, 834S, 834W, 855G, 856G, 857A, 857E, 857S, 857T, 857V, 869L, 869S, 871E, 871K, 873A, 873F, 873Y, 878A, 878G, 878K, 878Q, 878S, 878W, 909F, 924A, 926M, and 926T includes at least one substitution, where these positions are numbered with reference to Sequence ID No. 1956. In some embodiments, the acidic alpha-glucosidase is P78E / D87E / K176T / T266N / V536T / Q615D, P78E / D87E / T266N / S372T / A386Y / S777G, P78E / D87E / T266N / S372T / V536T, P78E / T266N / T763L, P78E / S372T / L390Q, D87E / T266N / S372T / T483S, D87E / S372T / S777G, A105T, A105W, Y125W, K129E, K129S, S136G, S136K, S 136V, G139E, T141S, A142G, F152S, F152W, K154R / F588L, I156C, I156L, V222C, V222P, N225D, T227A, A229C, T266N / S372T / V536T / Q615D / T763L / S777G, T266N / S372T / T924N, K267E, S372D, D401G, D493L, A496G, H499E, H499I, H499M, H499Q, L569H, L569Q, L569S, L569T, L569V, L569Y, A572G,A572S, I573C, I573D, I573H, I573M, I573Q, A574S, R577A, R577D, R577E, R577T, L579V, V580E, V580G, V580 W, K581G, K581H, K581T, R583C, R583G, T585Q, F588L, F588V, V589I / M663F, L628V, L629A, L629C, L629G, L62 9I, V631I, V631L, V631M, M663A, M663F, L669R, L670E, L670F, L670I, L670Q, L670R, L670S, L670T, L670V, L 670W, S671A, S671G, S671T, L691V, T692C, T692F, T692G, T692I, T692L, T692Y, L693F, L693I, L693Y, H706F, A708C, V710K, V710L, V710M, V710N, V710S, A711C, A711G, A711H, A711W, Y765W, Q768I, P779E, V797E, V797 F, V797I, V797L, V797M, V797W, T826G, A834G, A834H, A834S, A834W, E855G, S856G, L857A, L857E, L857S, L85 The sequence includes at least one substitution in a position or set of positions selected from 7T, L857V, I869L, I869S, L871E, L871K, R873A, R873F, R873Y, V878A, V878G, V878K, V878Q, V878S, V878W, L909F, T924A, V926M, and V926T, where these positions are numbered with reference to Sequence ID No. 1956.

[0017] The present invention provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to Sequence ID No. 2496, whose position is numbered with reference to Sequence ID No. 2496. In some embodiments, acid alpha-glucosidase is 246, 304, 313, 569, 569 / 588, 569 / 588 / 589 / 628 / 629 / 692 / 711, 569 / 588 / 678 / 692, 569 / 588 / 711 / 869 / 871 / 878, 569 / 588 / 878, 569 / 589 / 628 / 670 / 678 / 692 / 711 / 795 / 871, 569 / 589 / 628 / 670 / 692 / 711 , 569 / 589 / 628 / 692 / 711 / 795, 569 / 589 / 670, 569 / 589 / 670 / 678 / 692 / 711 / 795, 569 / 589 / 670 / 871, 569 / 589 / 678 / 871 / 878, 569 / 589 / 692 / 795 / 871 / 878, 569 / 589 / 711 / 871, 569 / 589 / 871, 569 / 628, 569 / 628 / 670, 569 / 628 / 670 / 678, 5 69 / 628 / 670 / 692 / 711 / 871, 569 / 628 / 670 / 711, 569 / 628 / 678 / 711, 569 / 628 / 692, 569 / 670, 569 / 670 / 678, 569 / 670 / 678 / 692 / 871, 569 / 670 / 692, 569 / 670 / 711, 569 / 670 / 711 / 871, 569 / 678 / 692 / 795, 569 / 678 / 869 / 878, 569 / 678 / 87 8, 569 / 692, 569 / 692 / 711, 569 / 692 / 711 / 869 / 871 / 878, 569 / 711, 569 / 711 / 795 / 871 / 878, 569 / 711 / 869 / 878, 569 / 711 / 871, 569 / 795, 569 / 871, 572 / 588 / 678 / 692 / 869 / 878, 572 / 588 / 795, 572 / 692 / 869 / 878, 572 / 692 / 878, 582, 584,585, 588 / 589 / 628 / 678, 589, 589 / 670 / 692 / 795 / 871, 589 / 670 / 795 / 871, 589 / 871, 628 / 629 / 692 / 871 / 878, 628 / 670, 628 / 670 / 692 / 711 / 795, 628 / 711 / 795, 628 / 871, 628 / 878, 629 / 869 / 878, 670 / 678, 670 / 678 / 692 / 871, 670 / 692 / 871, 678 / 692 / 711 / 869, 678 / 692 / 795 / 869, 678 / 692 / 869, 678 / 795 / 871 / 8 The position or set of positions selected from 78, 692, 692 / 711, 692 / 711 / 795 / 869, 692 / 711 / 795 / 869 / 871 / 878, 692 / 711 / 869 / 878, 692 / 711 / 871 / 878, 692 / 869, 692 / 869 / 871 / 878 / 916, 692 / 871, 711, 711 / 795 / 869 / 878, 711 / 869 / 878, 711 / 871, 795 / 878, 812, 871, and 878 includes at least one substitution, where these positions are numbered with reference to sequence number 2496. In some embodiments, the acid alpha-glucosidase is 246T, 304M, 313I, 313L, 569H / 589I / 628M / 670T / 678T / 692G / 711H / 795E / 871S, 569H / 628M, 569H / 628M / 670F / 678T, 569H / 628M / 670T, 569H / 628M / 670T / 692Y / 711H / 871K, 569H / 670F / 678T / 692G / 871S, 569H / 670T / 692G, 569H / 678T / 692G / 795E, 569H / 692G, 56 9H / 692Y, 569H / 711H / 795E / 871S / 878S, 569H / 711H / 869S / 878S, 569H / 7 11H / 871K, 569T, 569T / 588L, 569T / 588L / 589I / 628M / 629I / 692Y / 711H, 5 69T / 588L / 678T / 692Y, 569T / 588L / 711H / 869L / 871K / 878S, 569T / 588L / 878S, 569T / 589I / 628M / 670F / 692G / 711H, 569T / 589I / 678T / 871K / 878S,569T / 589I / 692G / 795E / 871K / 878S, 569T / 589I / 871S, 569T / 628M / 678T / 711H, 569T / 628M / 692Y, 569T / 670T / 678T / 692G / 871K, 569T / 670T / 711H / 871E 569T / 678T / 869S / 878S, 569T / 678T / 878S, 569T / 692Y, 569T / 692Y / 711H / 869L / 871K / 878A, 569T / 711H, 569T / 795E, 569T / 871K, 569T / 871S, 569Y / 589I / 628M / 670T / 692Y / 711H, 569Y / 589I / 628M / 692G / 711H / 795E, 569Y / 589I / 670F, 569Y / 589I / 670T / 678T / 692G / 711H / 795E, 569Y / 589I / 670T / 871K, 569Y / 589I / 711H / 871K、569Y / 628M / 670T / 692Y / 711H / 871S、569Y / 628M / 670T / 71 1H、569Y / 670F / 678T、569Y / 670T、569Y / 670T / 711H、569Y / 692Y / 711H、569Y / 711H, 569Y / 871E, 572S / 588L / 678T / 692G / 869L / 878A, 572S / 588L / 795E, 572S / 692G / 869S / 878S, 572S / 692G / 878S, 572S / 692L / 869S / 878S, 582T, 584E 585K, 588L / 589I / 628M / 678T, 589I, 589I / 670T / 692G / 795E / 871K, 589I / 670T / 795E / 871S, 589I / 871E, 628M / 629I / 692Y / 871S / 878S, 628M / 670F, 628M / 670T / 692G / 711H / 795E、628M / 711H / 795E、628M / 871S、628M / 878S、629I / 86 9L / 878S、670T / 678T、670T / 678T / 692Y / 871S、670T / 692G / 871K、678T / 692G / 711H / 869S, 678T / 692G / 795E / 869S, 678T / 692G / 869S, 678T / 795E / 871K / 878A, 692G / 711H, 692G / 711H / 795E / 869L / 871K / 878A, 692G / 711H / 795E / 869SThe positions or sets of positions selected from 692G / 711H / 869L / 878S, 692G / 711H / 869S / 878A, 692G / 711H / 871S / 878A, 692G / 869L / 871K / 878S / 916R, 692G / 869S, 692G / 871K, 692Y, 711H, 711H / 795E / 869S / 878S, 711H / 869S / 878S, 711H / 871K, 795E / 878S, 812E, 871K, 871S, and 878S include at least one substitution, where these positions are numbered with reference to sequence number 2496. In some embodiments, the acidic alpha-glucosidase is S246T, L304M, V313I, V313L, L569H / V589I / L628M / L670T / S678T / T692G / A711H / Q795E / L871S, L569H / L628M, L569H / L628M / L670F / S678T, L569H / L628M / L670T, L569H / L628M / L670T / T692Y / A71 1H / L871K, L569H / L670F / S678T / T692G / L871S, L569H / L670T / T692G, L569H / S678T / T692G / Q795E, L569H / T692G , L569H / T692Y, L569H / A711H / Q795E / L871S / V878S, L569H / A711H / I869S / V878S, L569H / A711H / L871K, L569T, L5 69T / F588L, L569T / F588L / V589I / L628M / L629I / T692Y / A711H, L569T / F588L / S678T / T692Y, L569T / F588L / A711 H / I869L / L871K / V878S, L569T / F588L / V878S, L569T / V589I / L628M / L670F / T692G / A711H, L569T / V589I / S678T / L 871K / V878S, L569T / V589I / T692G / Q795E / L871K / V878S, L569T / V589I / L871S, L569T / L628M / S678T / A711H, L56 9T / L628M / T692Y, L569T / L670T / S678T / T692G / L871K, L569T / L670T / A711H / L871E, L569T / S678T / I869S / V878S,L569T / S678T / V878S、L569T / T692Y、L569T / T692Y / A711H / I869L / L871K / V878A、L569T / A711H、L569T / Q795E、L569T / L871K、L569T / L871S、L569Y / V589I / L628M / L670T / T692Y / A711H、L569Y / V589I / L628M / T692G / A711H / Q795E、L569Y / V589I / L670F、L569Y / V589I / L670T / S678T / T692G / A711H / Q795E、L569Y / V589I / L670T / L871K、L569Y / V589I / A711H / L871K、L569Y / L628M / L670T / T692Y / A711H / L871S、L569Y / L628M / L670T / A711H、L569Y / L670F / S678T、L569Y / L670T、L569Y / L670T / A711H、L569Y / T692Y / A711H、L569Y / A711H、L569Y / L871E、A572S / F588L / S678T / T692G / I869L / V878A、A572S / F588L / Q795E、A572S / T692G / I869S / V878S、A572S / T692G / V878S、A572S / T692L / I869S / V878S、A582T、G584E、T585K、F588L / V589I / L628M / S678T、V589I、V589I / L670T / T692G / Q795E / L871K、V589I / L670T / Q795E / L871S、V589I / L871E、L628M / L629I / T692Y / L871S / V878S、L628M / L670F、L628M / L670T / T692G / A711H / Q795E、L628M / A711H / Q795E、L628M / L871S、L628M / V878S、L629I / I869L / V878S、L670T / S678T、L670T / S678T / T692Y / L871S、L670T / T692G / L871K、S678T / T692G / A711H / I869S、S678T / T692G / Q795E / I869S、S678T / T692G / I869S、S678T / Q795E / L871K / V878A、T692G / A711H、T692G / A711H / Q795E / I869L / L871K / V878A、T692G / A711H / Q795E / I869S、T692G / A711H / I869L / V878S、T692G / A711H / I869S / V878A、T692G / A711H / L871S / V878A、T692G / I869L / L871K / V878S / S916R、T692G / I869S、T692G / L87、 The acidic alpha-glucosidase includes at least one substitution in a position or set of positions selected from 1K, T692Y, A711H, A711H / Q795E / I869S / V878S, A711H / I869S / V878S, A711H / L871K, Q795E / V878S, A812E, L871K, L871S, and V878S, where these positions are numbered with reference to Sequence ID No. 2496. In some embodiments, the acidic alpha-glucosidase includes at least one substitution in a position or set of positions selected from 60 / 589, 307, 313, 584, and 810, where these positions are numbered with reference to Sequence ID No. 2496. In some embodiments, the acidic alpha-glucosidase includes at least one substitution in a position or set of positions selected from 60V / 589A, 307T, 313T, 584C, and 810V, where these positions are numbered with reference to Sequence ID No. 2496. In some embodiments, acid alpha-glucosidase is 60 / 589, 246, 304, 307, 313, 569, 569 / 588, 569 / 588 / 589 / 628 / 629 / 692 / 711, 569 / 588 / 678 / 692, 569 / 588 / 711 / 869 / 871 / 878, 569 / 588 / 878, 569 / 589 / 628 / 670 / 678 / 692 / 711 / 795 / 871, 569 / 589 / 628 / 670 / 692 / 711, 569 / 589 / 628 / 692 / 711 / 795, 569 / 589 / 670, 569 / 589 / 670 / 678 / 692 / 711 / 795, 569 / 589 / 670 / 871, 569 / 589 / 678 / 871 / 878, 569 / 589 / 692 / 795 / 871 / 878, 569 / 589 / 711 / 871, 569 / 589 / 871, 569 / 628, 569 / 628 / 670, 569 / 628 / 670 / 678, 569 / 628 / 670 / 692 / 711 / 871, 569 / 628 / 670 / 711, 569 / 628 / 678 / 711, 569 / 628 / 692, 569 / 670,569 / 670 / 678, 569 / 670 / 678 / 692 / 871, 569 / 670 / 692, 569 / 670 / 711, 569 / 670 / 711 / 871, 569 / 678 / 692 / 795, 569 / 678 / 869 / 878, 569 / 678 / 878, 569 / 692, 569 / 692 / 711, 569 / 692 / 711 / 869 / 871 / 878, 569 / 711, 569 / 711 / 795 / 871 / 878, 569 / 711 / 869 / 8 78, 569 / 711 / 871, 569 / 795, 569 / 871, 572 / 588 / 678 / 692 / 869 / 878, 572 / 588 / 795, 572 / 692 / 869 / 878, 572 / 692 / 878, 582, 584, 585, 588 / 589 / 628 / 678, 589, 589 / 670 / 692 / 795 / 871, 589 / 670 / 795 / 871, 589 / 871, 628 / 629 / 692 / 871 / 878, 628 / 670, 628 / 670 / 692 / 711 / 795, 628 / 711 / 795, 628 / 871, 628 / 878, 629 / 869 / 878, 670 / 678, 670 / 678 / 692 / 871, 670 / 692 / 871, 678 / 692 / 711 / 869, 678 / 692 / 795 / 869, 678 / 692 / 869, 678 / 795 / 871 / 878, 692, 692 / 711, 692 / 711 / 795 / 869, 692 / 711 / 795 / 869 / 871 / The position or set of positions selected from 878, 692 / 711 / 869 / 878, 692 / 711 / 871 / 878, 692 / 869, 692 / 869 / 871 / 878 / 916, 692 / 871, 711, 711 / 795 / 869 / 878, 711 / 869 / 878, 711 / 871, 795 / 878, 810, 812, 871, and 878 includes at least one substitution, where these positions are numbered with reference to sequence number 2496. In some embodiments, the acid alpha-glucosidase is 60V / 589A, 246T, 304M, 307T, 313I, 313L, 313T, 569H / 589I / 628M / 670T / 678T / 692G / 711H / 795E / 871S, 569H / 628M, 569H / 628M / 670F / 678T, 569H / 628M / 670T, 569H / 628M / 670T / 692Y / 711H / 871K,569H / 670F / 678T / 692G / 871S, 569H / 670T / 692G, 569H / 678T / 692G / 795E, 569H / 692G, 569H / 692Y, 569H / 711H / 795E / 871S / 878S, 569H / 711H / 869S / 878S 569H / 711H / 871K, 569T, 569T / 588L, 569T / 588L / 589I / 628M / 629I / 692Y / 711H, 569T / 588L / 678T / 692Y, 569T / 588L / 711H / 869L / 871K / 878S, 569T / 588 L / 878S, 569T / 589I / 628M / 670F / 692G / 711H, 569T / 589I / 678T / 871K / 878S, 569T / 589I / 692G / 795E / 871K / 878S, 569T / 589I / 871S, 569T / 628M / 678T / 71 1H, 569T / 628M / 692Y, 569T / 670T / 678T / 692G / 871K, 569T / 670T / 711H / 871E, 569T / 678T / 869S / 878S, 569T / 678T / 878S, 569T / 692Y, 569T / 692Y / 711H / 86 9L / 871K / 878A, 569T / 711H, 569T / 795E, 569T / 871K, 569T / 871S, 569Y / 589I / 628M / 670T / 692Y / 711H, 569Y / 589I / 628M / 692G / 711H / 795E, 569Y / 589I / 6 70F, 569Y / 589I / 670T / 678T / 692G / 711H / 795E, 569Y / 589I / 670T / 871K, 569Y / 589I / 711H / 871K, 569Y / 628M / 670T / 692Y / 711H / 871S, 569Y / 628M / 670T / 7 11H、569Y / 670F / 678T、569Y / 670T、569Y / 670T / 711H、569Y / 692Y / 711H、569 Y / 711H、569Y / 871E、572S / 588L / 678T / 692G / 869L / 878A、572S / 588L / 795E、 572S / 692G / 869S / 878S, 572S / 692G / 878S, 572S / 692L / 869S / 878S, 582T, 584C, 584E, 585K, 588L / 589I / 628M / 678T, 589I, 589I / 670T / 692G / 795E / 871K589I / 670T / 795E / 871S, 589I / 871E, 628M / 629I / 692Y / 871S / 878S, 628M / 670F, 628M / 67 0T / 692G / 711H / 795E, 628M / 711H / 795E, 628M / 871S, 628M / 878S, 629I / 869L / 878S, 670T / 678T, 670T / 678T / 692Y / 871S, 670T / 692G / 871K, 678T / 692G / 711H / 869S, 678T / 692G / 79 5E / 869S, 678T / 692G / 869S, 678T / 795E / 871K / 878A, 692G / 711H, 692G / 711H / 795E / 869L / 871K / 878A, 692G / 711H / 795E / 869S, 692G / 711H / 869L / 878S, 692G / 711H / 869S / 878A, 69 2G / 711H / 871S / 878A, 692G / 869L / 871K / 878S / 916R, 692G / 869S, 692G / 871K, 692Y, 711H, The position or set of positions selected from 711H / 795E / 869S / 878S, 711H / 869S / 878S, 711H / 871K, 795E / 878S, 810V, 812E, 871K, 871S, and 878S includes at least one substitution, where these positions are numbered with reference to sequence number 2496. In some embodiments, the acidic alpha-glucosidase is A60V / V589A, S246T, L304M, S307T, V313I, V313L, V313T, L569H / V589I / L628M / L670T / S678T / T692G / A711H / Q795E / L871S, L569H / L628M, L569H / L628M / L670F / S678T, L569H / L628M / L670T, L569H / L628M / L670T / T6 92Y / A711H / L871K, L569H / L670F / S678T / T692G / L871S, L569H / L670T / T692G, L569H / S678T / T692G / Q795E, L569H / T692G, L 569H / T692Y, L569H / A711H / Q795E / L871S / V878S, L569H / A711H / I869S / V878S, L569H / A711H / L871K, L569T, L569T / F588L,L569T / F588L / V589I / L628M / L629I / T692Y / A711H、L569T / F588L / S678T / T 692Y、L569T / F588L / A711H / I869L / L871K / V878S、L569T / F588L / V878S、L5 69T / V589I / L628M / L670F / T692G / A711H, L569T / V589I / S678T / L871K / V878S, L569T / V589I / T692G / Q795E / L871K / V878S, L569T / V589I / L871S, L569 T / L628M / S678T / A711H、L569T / L628M / T692Y、L569T / L670T / S678T / T692G / L871K、L569T / L670T / A711H / L871E、L569T / S678T / I869S / V878S、L569T / S678T / V878S, L569T / T692Y, L569T / T692Y / A711H / I869L / L871K / V878A, L569T / A711H, L569T / Q795E, L569T / L871K, L569T / L871S, L569Y / V589I / L62 8M / L670T / T692Y / A711H, L569Y / V589I / L628M / T692G / A711H / Q795E, L569Y / V589I / L670F, L569Y / V589I / L670T / S678T / T692G / A711H / Q795E, L569Y / V589I / L670T / L871K、L569Y / V589I / A711H / L871K、L569Y / L628M / L670T / T692Y / A711H / L871S、L569Y / L628M / L670T / A711H、L569Y / L670F / S678T、L 569Y / L670T、L569Y / L670T / A711H、L569Y / T692Y / A711H、L569Y / A711H、L5 69Y / L871E、A572S / F588L / S678T / T692G / I869L / V878A、A572S / F588L / Q79 5E、A572S / T692G / I869S / V878S、A572S / T692G / V878S、A572S / T692L / I869 S / V878S、A582T、G584C、G584E、T585K、F588L / V589I / L628M / S678T、V589I、V589I / L670T / T692G / Q795E / L871K, V589I / L670T / Q795E / L871S, V589I / L871E, L628M / L629I / T692Y / L87 1S / V878S, L628M / L670F, L628M / L670T / T692G / A711H / Q795E, L628M / A711H / Q795E, L628M / L871S, L628M / , V878S, L629I / I869L / V878S, L670T / S678T, L670T / S678T / T692Y / L871S, L670T / T 692G / L871K, S678T / T692G / A711H / I869S, S678T / T692G / Q795E / I869S, S678T / T69 2G / I869S, S678T / Q795E / L871K / V878A, T692G / A711H, T692G / A711H / Q795E / I869 L / L871K / V878A, T692G / A711H / Q795E / I869S, T692G / A711H / I869L / V878S, T692G / The positions or sets of positions selected from A711H / I869S / V878A, T692G / A711H / L871S / V878A, T692G / I869L / L871K / V878S / S916R, T692G / I869S, T692G / L871K, T692Y, A711H, A711H / Q795E / I869S / V878S, A711H / I869S / V878S, A711H / L871K, Q795E / V878S, L810V, A812E, L871K, L871S, and V878S include at least one substitution, where these positions are numbered with reference to sequence number 2496.

[0018] The present invention provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to sequence number 2880, whose position is numbered with reference to sequence number 2880. In some embodiments, acid alpha-glucosidase is 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 522 / 527 / 551 / 670 / 727 / 750 / 830 / 842 / 871 / 883 / 894 / 932, 24 / 28 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 522 / 527 / 551 / 569 / 727 / 830 / 842 / 871 / 883 / 8 94 / 913, 24 / 28 / 50 / 135 / 150 / 437 / 522 / 527 / 871 / 883 / 894 / 932, 24 / 28 / 62 / 522 / 569 / 932, 24 / 28 / 437 / 486 / 527, 24 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 267 / 437 / 486 / 522 / 527 / 551 / 711 / 727 / 750 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 5 0 / 78 / 87 / 135 / 150 / 267 / 486 / 522 / 527 / 551 / 670 / 727 / 750 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 50 / 486 / 527 / 711 / 727, 24 / 62 / 87 / 486 / 727, 24 / 62 / 727 / 830 / 932, 24 / 87 / 135 / 522 / 670 / 711 / 830 / 842 / 913, 24 / 150 / 522 / 527 / 727 / 88 3 / 894, 24 / 527 / 727 / 842 / 871 / 883 / 913 / 932, 24 / 670 / 727 / 750 / 842 / 871, 28, 28 / 50 / 78 / 87 / 135 / 266 / 267 / 437 / 486 / 527 / 551 / 670 / 727 / 750 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 28 / 50 / 522 / 527 / 711 / 727 / 871, 28 / 62, 28 / 62 / 267 / 932,28 / 437 / 527 / 871、28 / 522 / 527 / 569 / 711 / 830 / 894、28 / 727、28 / 727 / 871、29 / 39 / 50 / 62 / 65 / 78 / 87 / 135 / 150 / 437 / 551 / 569 / 670 / 727 / 750 / 830 / 842 / 883 / 894 / 932、29 / 62 / 437 / 527、29 / 78 / 87 / 150 / 527 / 727、29 / 78 / 135 / 727 / 830、29 / 87、29 / 135 / 150 / 527 / 670 / 727 / 883、29 / 150 / 267 / 727 / 750 / 871 / 883 / 932、29 / 150 / 437 / 727、29 / 522 / 670 / 711 / 871、29 / 670 / 932、39 / 50、39 / 727 / 750 / 932、50 / 135 / 150 / 932、50 / 437 / 522 / 527、50 / 711、50 / 727 / 750 / 883 / 894、62、62 / 87 / 150、62 / 87 / 150 / 727、62 / 135 / 522 / 711 / 727 / 750 / 842 / 871 / 894、62 / 437、62 / 437 / 727、78 / 87 / 486 / 527 / 670 / 727 / 750 / 830 / 842 / 871 / 913 / 932、87 / 750、89 / 109 / 527 / 678 / 727 / 842、89 / 109 / 678 / 727 / 736 / 812 / 878、89 / 109 / 727 / 932、89 / 109 / 932、89 / 527、89 / 527 / 678 / 692 / 736 / 842 / 878 / 932、89 / 527 / 678 / 932、89 / 527 / 727 / 812 / 860、89 / 678 / 692 / 736 / 932、89 / 678 / 812 / 878、89 / 842 / 878、109 / 527 / 678 / 812、109 / 678 / 692 / 842 / 860 / 878 / 932、109 / 678 / 727 / 860、109 / 678 / 736 / 812 / 878、109 / 678 / 812、109 / 678 / 842 / 878、109 / 692 / 727 / 736 / 812、109 / 692 / 727 / 812 / 842 / 860、109 / 727 / 860 / 878、109 / 736 / 932、109 / 812、109 / 842、109 / 932、135 / 670 / 727、135 / 711 / 750 / 932、150 / 527 / 842 / 871 / 913、150 / 871 / 932、150 / 883 / 932、267 / 527 / 727、403 / 527 / 678 / 692 / 736 / 812 / 842 / 860, 437 / 522 / 527 / 670 / 871, 437 / 750 / 830 / 932, 522, 522 / 527 / 569 / 727, 522 / 830, 527, 527 / 678 / 692 / 727 / 736 / 878, 527 / 678 / 692 / 812 / 932, 527 / 692 / 727 / 736 / 812, 527 / 692 / 727 / 736 / 842 / 860 / 878, 527 / 727 / 736, 527 / 736 / 932, 5 The acidic alpha-glucosidase includes at least one substitution in a position or set of positions selected from 27 / 812, 670 / 711 / 871, 670 / 830 / 871, 678 / 692 / 727 / 812 / 842, 678 / 692 / 812, 678 / 812, 678 / 860 / 878, 678 / 913, 678 / 932, 692 / 727 / 736 / 842 / 913, 692 / 812, 727, 727 / 932, 871, and 878 / 932, where these positions are numbered with reference to Sequence ID No. 2880. In some embodiments, the acidic alpha-glucosidase includes at least one substitution in a position or set of positions selected from __, where these positions are numbered with reference to Sequence ID No. 2880. In some embodiments, acid alpha-glucosidase is 24L / 28L / 29L / 39P / 50Q / 62A / 78P / 87D / 135S / 150T / 266T / 267R / 522E / 527N / 551V / 670L / 727S / 750A / 830Q / 842G / 871L / 883R / 894Q / 932S, 24L / 28L / 39P / 50Q / 62A / 78P / 87D / 135S / 150T / 266T / 267R / 522E / 527N / 551V / 569L / 727S / 830Q / 842G / 871L / 883R / 89 4Q / 913V, 24L / 28L / 50Q / 135S / 150T / 437A / 522E / 527N / 871L / 883R / 894 Q / 932S, 24L / 28L / 62A / 522E / 569L / 932S, 24L / 28L / 437A / 486T / 527N, 2 4L / 29L / 39P / 50Q / 62A / 78P / 87D / 135S / 150T / 267R / 437A / 486T / 522E / 5 27N / 551V / 711A / 727S / 750A / 830Q / 842G / 871L / 883R / 894Q / 913V / 932S,24L / 50Q / 78P / 87D / 135S / 150T / 267R / 486T / 522E / 527N / 551V / 670L / 727S / 750A / 830Q / 842G / 871L / 883R / 894Q / 913V / 932S、24L / 50Q / 486T / 527N / 711A / 727S、24L / 62A / 87D / 486T / 727S、24L / 62A / 727S / 830Q / 932S、24L / 87D / 135S / 522E / 670L / 711A / 830Q / 842G / 913V、24L / 150T / 522E / 527N / 727S / 883R / 8 94Q, 24L / 527N / 727S / 842G / 871L / 883R / 913V / 932S, 24L / 670L / 727S / 750A / 842G / 871L, 28L, 28L / 50Q / 78P / 87D / 135S / 266T / 267R / 437A / 486T / 527N / 55 1V / 670L / 727S / 750A / 830Q / 842G / 871L / 883R / 894Q / 913V / 932S, 28L / 50Q / 522E / 527N / 711A / 727S / 871L, 28L / 62A, 28L / 62A / 267R / 932S, 28L / 437A / 527 N / 871L, 28L / 522E / 527N / 569L / 711A / 830Q / 894Q, 28L / 727S, 28L / 727S / 871L, 29L / 39P / 50Q / 62A / 65R / 78P / 87D / 135S / 150T / 437A / 551V / 569L / 670L / 72 7S / 750A / 830Q / 842G / 883R / 894Q / 932S, 29L / 62A / 437A / 527N, 29L / 78P / 87D / 150T / 527N / 727S, 29L / 78P / 135S / 727S / 830Q, 29L / 87D, 29L / 135S / 150T / 5 27N / 670L / 727S / 883R, 29L / 150T / 267R / 727S / 750A / 871L / 883R / 932S, 29L / 150T / 437A / 727S, 29L / 522E / 670L / 711A / 871L, 29L / 670L / 932S, 39P / 50Q, 3 9P / 727S / 750A / 932S, 50Q / 135S / 150T / 932S, 50Q / 437A / 522E / 527N, 50Q / 711A, 50Q / 727S / 750A / 883R / 894Q, 62A, 62A / 87D / 150T, 62A / 87D / 150T / 727S62A / 135S / 522E / 711A / 727S / 750A / 842G / 871L / 894Q、62A / 437A、62A / 437A / 727S、78P / 87D / 486T / 527N / 670L / 727S / 750A / 830Q / 842G / 871L / 913V / 932S、87D / 750A、89R / 109D / 527N / 678T / 727S / 842G、89R / 109D / 678T / 727S / 736M / 812E / 878S、89R / 109D / 727S / 932S、89R / 109D / 932S、89R / 527N、89R / 527N / 678T / 692G / 736M / 842G / 878S / 932S、89R / 527N / 678T / 932S、89R / 527N / 727S / 812E / 860F、89R / 678T / 692G / 736M / 932S、89R / 678T / 812E / 878S、89R / 842G / 878S、109D / 527N / 678T / 812E、109D / 678T / 692G / 842G / 860F / 878S / 932S、109D / 678T / 727S / 860F、109D / 678T / 736M / 812E / 878S、109D / 678T / 812E、109D / 678T / 842G / 878S、109D / 692G / 727S / 736M / 812E、109D / 692G / 727S / 812E / 842G / 860F、109D / 727S / 860F / 878S、109D / 736M / 932S、109D / 812E、109D / 842G、109D / 932S、135S / 670L / 727S、135S / 711A / 750A / 932S、150T / 527N / 842G / 871L / 913V、150T / 871L / 932S、150T / 883R / 932S、267R / 527N / 727S、403H / 527N / 678T / 692G / 736M / 812E / 842G / 860F、437A / 522E / 527N / 670L / 871L、437A / 750A / 830Q / 932S、522E、522E / 527N / 569L / 727S、522E / 830Q、527N、527N / 678T / 692G / 727S / 736M / 878S、527N / 678T / 692G / 812E / 932S、527N / 692G / 727S / 736M / 812E、527N / 692G / 727S / 736M / 842G / 860F / 878S、527N / 727S / 736M、527N / 736M / 932S、527N / 812E、670L / 711A / 871L、670L / 830Q / 871L、678T / 692G / 727S / 812E / 842G、678T / 692G / 812E、678T / 812E、678T / 860F / 878S、678T / 913V、678T / 932S、692G / 727S / 736M / 842G / 91、 The substitution includes at least one substitution in a position or set of positions selected from 3V, 692G / 812E, 727S, 727S / 932S, 871L, and 878S / 932S, these positions being numbered with reference to sequence number 2880. In some embodiments, the acidic alpha-glucosidase is W24L / S28L / T29L / Q39P / V50Q / L62A / E78P / E87D / Q135S / S150T / N266T / K267R / V522E / R527N / A551V / T670L / W727S / P750A / K830Q / S842G / E871L / H883R / G894Q / A932S, W24L / S28L / Q39P / V50Q / L62A / E78P / E87D / Q135S / S150T / N266T / K 267R / V522E / R527N / A551V / T569L / W727S / K830Q / S842G / E871L / H883R / G894Q / R913V, W24L / S28L / V50Q / Q135S / S150T / G437A / V522 E / R527N / E871L / H883R / G894Q / A932S, W24L / S28L / L62A / V522E / T569L / A932S, W24L / S28L / G437A / E486T / R527N, W24L / T29L / Q39P / V50Q / L62A / E78P / E87D / Q135S / S150T / K267R / G437A / E486T / V522E / R527N / A551V / H711A / W727S / P750A / K830Q / S842G / E871L / H883 R / G894Q / R913V / A932S, W24L / V50Q / E78P / E87D / Q135S / S150T / K267R / E486T / V522E / R527N / A551V / T670L / W727S / P750A / K830Q / S8 42G / E871L / H883R / G894Q / R913V / A932S, W24L / V50Q / E486T / R527N / H711A / W727S, W24L / L62A / E87D / E486T / W727S, W24L / L62A / W72 7S / K830Q / A932S, W24L / E87D / Q135S / V522E / T670L / H711A / K830Q / S842G / R913V, W24L / S150T / V522E / R527N / W727S / H883R / G894Q,W24L / R527N / W727S / S842G / E871L / H883R / R913V / A932S、W24L / T670L / W72 7S / P750A / S842G / E871L、S28L、S28L / V50Q / E78P / E87D / Q135S / N266T / K26 7R / G437A / E486T / R527N / A551V / T670L / W727S / P750A / K830Q / S842G / E871L / H883R / G894Q / R913V / A932S, S28L / V50Q / V522E / R527N / H711A / W727S / E 871L, S28L / L62A, S28L / L62A / K267R / A932S, S28L / G437A / R527N / E871L, S28L / V522E / R527N / T569L / H711A / K830Q / G894Q, S28L / W727S, S28L / W727S / E871L、T29L / Q39P / V50Q / L62A / G65R / E78P / E87D / Q135S / S150T / G437A / A551V / T569L / T670L / W727S / P750A / K830Q / S842G / H883R / G894Q / A932S、T29 L / L62A / G437A / R527N, T29L / E78P / E87D / S150T / R527N / W727S, T29L / E78P / Q135S / W727S / K830Q, T29L / E87D, T29L / Q135S / S150T / R527N / T670L / W72 7S / H883R, T29L / S150T / K267R / W727S / P750A / E871L / H883R / A932S, T29L / S150T / G437A / W727S, T29L / V522E / T670L / H711A / E871L, T29L / T670L / A932 S, Q39P / V50Q, Q39P / W727S / P750A / A932S, V50Q / Q135S / S150T / A932S, V50Q / G437A / V522E / R527N, V50Q / H711A, V50Q / W727S / P750A / H883R / G894Q, L 62A、L62A / E87D / S150T、L62A / E87D / S150T / W727S、L62A / Q135S / V522E / H7 11A / W727S / P750A / S842G / E871L / G894Q、L62A / G437A、L62A / G437A / W727S、E78P / E87D / E486T / R527N / T670L / W727S / P750A / K830Q / S842G / E871L / R913V / A932S、E87D / P750A、A89R / L109D / R527N / S678T / W727S / S842G、A89R / L109D / S678T / W727S / L736M / A812E / V878S、A89R / L109D / W727S / A932S、A89R / L109D / A932S、A89R / R527N、A89R / R527N / S678T / T692G / L736M / S842G / V878S / A932S、A89R / R527N / S678T / A932S、A89R / R527N / W727S / A812E / L860F、A89R / S678T / T692G / L736M / A932S、A89R / S678T / A812E / V878S、A89R / S842G / V878S、L109D / R527N / S678T / A812E、L109D / S678T / T692G / S842G / L860F / V878S / A932S、L109D / S678T / W727S / L860F、L109D / S678T / L736M / A812E / V878S、L109D / S678T / A812E、L109D / S678T / S842G / V878S、L109D / T692G / W727S / L736M / A812E、L109D / T692G / W727S / A812E / S842G / L860F、L109D / W727S / L860F / V878S、L109D / L736M / A932S、L109D / A812E、L109D / S842G、L109D / A932S、Q135S / T670L / W727S、Q135S / H711A / P750A / A932S、S150T / R527N / S842G / E871L / R913V、S150T / E871L / A932S、S150T / H883R / A932S、K267R / R527N / W727S、R403H / R527N / S678T / T692G / L736M / A812E / S842G / L860F、G437A / V522E / R527N / T670L / E871L、G437A / P750A / K830Q / A932S、V522E、V522E / R527N / T569L / W727S、V522E / K830Q、R527N、R527N / S678T / T692G / W727S / L736M / V878S、R527N / S678T / T692G / A812E / A932S, R527N / T692G / W727S / L736M / A812E, R527N / T692G / W727S / L736M / S842G / L860F / V878S, R527N / W727S / L736M, R527N / L736M / A932S, R527N / A812E, T670L / H711A / E871L, T670L / K830Q / E871L, S678T / T692G / W727S / A812E / S842G, The system includes at least one substitution in a position or set of positions selected from S678T / T692G / A812E, S678T / A812E, S678T / L860F / V878S, S678T / R913V, S678T / A932S, T692G / W727S / L736M / S842G / R913V, T692G / A812E, W727S, W727S / A932S, E871L, and V878S / A932S, where these positions are numbered with reference to sequence number 2880. In some embodiments, acid alpha-glucosidase is 62 / 87 / 150, 89 / 109 / 527 / 678 / 727 / 842, 89 / 109 / 678 / 727 / 736 / 812 / 878, 89 / 109 / 932, 89 / 527 / 678 / 692 / 736 / 842 / 878 / 932, 89 / 527 / 727 / 8 12 / 860, 89 / 678 / 692 / 736 / 932, 89 / 678 / 812 / 878, 109 / 527 / 678 / 812, 109 / 678 / 692 / 842 / 860 / 878 / 932, 109 / 678 / 736 / 812 / 878, 109 / 678 / 812, 109 / 692 / 727 / 736 / 812, 109 / 692 / 727 / 812 / 842 / 860, 109 / 736 / 932, 109 / 812, 109 / 842, 109 / 932, 403 / 527 / 678 / 692 / 736 / 812 / 842 / 860, 522 / 830, 527 / 678 / 692 / 727 / 736 / 878, 527 / 678 / 692 / 812 / 932 , 527 / 692 / 727 / 736 / 812, 527 / 692 / 727 / 736 / 842 / 860 / 878, 527 / 727 / 736, 527 / 736 / 932, 527 / 812, 678 / 692 / 727 / 812 / 842, 678 / 692 / 812, 678 / 812, 692 / 727 / 736 / 842 / 913,The substitution includes at least one substitution in a position or set of positions selected from 692 / 812, where these positions are numbered with reference to Sequence ID No. 2880. In some embodiments, the acid alpha-glucosidase is 62A / 87D / 150T, 89R / 109D / 527N / 678T / 727S / 842G, 89R / 109D / 678T / 727S / 736M / 812E / 878S, 89R / 109D / 932S, 89R / 527N / 678T / 692G / 736M / 842G / 878S / 932S, 89R / 527N / 727S / 812E / 860F, 89R / 678T / 692G / 736M / 932S, 89R / 678T / 812E / 878S, 109D / 527N / 678T / 812E, 109D / 678T / 692G / 842G / 860F / 878S / 932S, 109D / 678T / 73 6M / 812E / 878S, 109D / 678T / 812E, 109D / 692G / 727S / 736M / 812E, 109D / 692G / 727S / 812E / 842G / 860F, 109D / 736M / 932S , 109D / 812E, 109D / 842G, 109D / 932S, 403H / 527N / 678T / 692G / 736M / 812E / 842G / 860F, 522E / 830Q, 527N / 678T / 692G / 727S / 736M / 878S, 527N / 678T / 692G / 812E / 932S, 527N / 692G / 727S / 736M / 812E, 527N / 692G / 727S / 736M / 842G / 860F / 87 The substitution includes at least one substitution in a position or set of positions selected from 8S, 527N / 727S / 736M, 527N / 736M / 932S, 527N / 812E, 678T / 692G / 727S / 812E / 842G, 678T / 692G / 812E, 678T / 812E, 692G / 727S / 736M / 842G / 913V, and 692G / 812E, where these positions are numbered with reference to sequence number 2880. In some embodiments, the acidic alpha-glucosidases are L62A / E87D / S150T, A89R / L109D / R527N / S678T / W727S / S842G, A89R / L109D / S678T / W727S / L736M / A812E / V878S, A89R / L109D / A932S,A89R / R527N / S678T / T692G / L736M / S842G / V878S / A932S, A89R / R527N / W727S / A812E / L860F, A89R / S678T / T692G / L736M / A 932S, A89R / S678T / A812E / V878S, L109D / R527N / S678T / A812E, L109D / S678T / T692G / S842G / L860F / V878S / A932S, L109D / , S678T / L736M / A812E / V878S, L109D / S678T / A812E, L109D / T692G / W727S / L736M / A812E, L109D / T692G / W727S / A812E / S842G / L860F, L109D / L736M / A932S, L109D / A812E, L109D / S842G, L109D / A932S, R403H / R527N / S678T / T692G / L736M / A812E / S842G / L860F, V522E / K830Q, R527N / S678T / T692G / W727S / L736M / V878S, R527N / S678T / T692G / A812E / A932S, R527N / T692G / W727S / L736M / A812E, R527N / T692G / W727S / L736M / S842G / L860F / V878S, R527N / W727S / L736M, R527N / L736M / A932S, R527N / A812E, S678T / T692G / W727S / A812E / The substitution includes at least one substitution in a position or set of positions selected from S842G, S678T / T692G / A812E, S678T / A812E, T692G / W727S / L736M / S842G / R913V, and T692G / A812E, where these positions are numbered with reference to sequence number 2880.

[0019] The present invention provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 3104, with the position numbered with reference to SEQ ID NO: 3104. In some embodiments, acid alpha-glucosidase is 62, 62 / 89 / 830, 62 / 248 / 678 / 830 / 878 / 932, 62 / 678, 62 / 678 / 785, 62 / 678 / 830, 62 / 678 / 830 / 860, 62 / 678 / 830 / 860 / 871 / 878 / 932, 62 / 678 / 830 / 860 / 878, 62 / 678 / 830 / 860 / 878 / 932, 62 / 678 / 830 / 860 / 932, 62 / 678 / 830 / 871, 62 / 678 / 830 / 871 / 932, 62 / 678 / 830 / 878 / 932, 62 / 678 / 830 / 932, 62 / 678 / 860, 62 / 678 / 860 / 878, 62 / 678 / 860 / 932, 62 / 678 / 871, 62 / 678 / 871 / 932, 62 / 678 / 878 / 932, 62 / 678 / 932, 62 / 830, 62 / 830 / 860, 62 / 830 / 860 / 871 / 873, 62 / 830 / 860 / 878 / 932, 62 / 830 / 860 / 932, 62 / 830 / 871 / 932, 62 / 830 / 878, 62 / 830 / 932, 62 / 833 / 860 / 932, 62 / 860, 62 / 860 / 871, 62 / 860 / 871 / 878, 62 / 860 / 871 / 932 , includes at least one substitution in a position or set of positions selected from 62 / 860 / 878 / 932, 62 / 860 / 932, 62 / 871 / 878 / 932, 62 / 871 / 932, 62 / 878, 62 / 878 / 932, 62 / 932, 678, 678 / 830 / 932, 678 / 932, and 860 / 932, where these positions are numbered with reference to Sequence ID No. 3104. In some embodiments, the acid alpha-glucosidase is 62A, 62A / 89D / 830Q,62A / 248H / 678T / 830Q / 878S / 932S, 62A / 678T, 62A / 678T / 785Q, 62A / 678T / 830Q, 62A / 678T / 830Q / 860F, 62A / 678T / 830Q / 860F / 8 71L / 878S / 932S, 62A / 678T / 830Q / 860F / 878S, 62A / 678T / 830Q / 860F / 878S / 932S, 62A / 678T / 830Q / 860F / 932S, 62A / 678T / 830Q / 871L, 62A / 678T / 830Q / 871L / 932S, 62A / 678T / 830Q / 878S / 932S, 62A / 678T / 830Q / 932S, 62A / 678T / 860F, 62A / 678T / 860F / 878S, 62A / 678T / 860F / 932S, 62A / 678T / 871L, 62A / 678T / 871L / 932S, 62A / 678T / 878S / 932S, 62A / 678T / 878S / 932T, 62A / 678T / 932S, 6 2A / 830Q, 62A / 830Q / 860F, 62A / 830Q / 860F / 871L / 873H, 62A / 830Q / 860F / 878S / 932S, 62A / 830Q / 860F / 932S, 62A / 830Q / 871L / 93 2S, 62A / 830Q / 878S, 62A / 830Q / 932S, 62A / 833I / 860F / 932S, 62A / 860F, 62A / 860F / 871L, 62A / 860F / 871L / 878S, 62A / 860F / 871L The positions or sets of positions selected from / 932S, 62A / 860F / 878S / 932S, 62A / 860F / 932S, 62A / 871L / 878S / 932S, 62A / 871L / 932S, 62A / 878S, 62A / 878S / 932S, 62A / 932S, 678T, 678T / 830Q / 932S, 678T / 932S, and 860F / 932S include at least one substitution, where these positions are numbered with reference to sequence number 3104. In some embodiments, the acidic alpha-glucosidase is L62A, L62A / A89D / K830Q, L62A / Y248H / S678T / K830Q / V878S / A932S, L62A / S678T, L62A / S678T / P785Q, L62A / S678T / K830Q, L62A / S678T / K830Q / L860F,L62A / S678T / K830Q / L860F / E871L / V878S / A932S, L62A / S678T / K830Q / L860F / V878S, L62A / S678T / K830Q / L860F / V878S / A932S, L62A / S6 78T / K830Q / L860F / A932S, L62A / S678T / K830Q / E871L, L62A / S678T / K830Q / E871L / A932S, L62A / S678T / K830Q / V878S / A932S, L62A / S678T / K830Q / A932S, L62A / S678T / L860F, L62A / S678T / L860F / V878S, L62A / S678T / L860F / A932S, L62A / S678T / E871L, L62A / S678T / E871L / A9 32S, L62A / S678T / V878S / A932S, L62A / S678T / V878S / A932T, L62A / S678T / A932S, L62A / K830Q, L62A / K830Q / L860F, L62A / K830Q / L860F / E 871L / R873H, L62A / K830Q / L860F / V878S / A932S, L62A / K830Q / L860F / A932S, L62A / K830Q / E871L / A932S, L62A / K830Q / V878S, L62A / K830 Q / A932S, L62A / M833I / L860F / A932S, L62A / L860F, L62A / L860F / E871L, L62A / L860F / E871L / V878S, L62A / L860F / E871L / A932S, L62A / L86 The sequence includes at least one substitution in a position or set of positions selected from 0F / V878S / A932S, L62A / L860F / A932S, L62A / E871L / V878S / A932S, L62A / E871L / A932S, L62A / V878S, L62A / V878S / A932S, L62A / A932S, S678T, S678T / K830Q / A932S, S678T / A932S, and L860F / A932S, where these positions are numbered with reference to sequence number 3104.

[0020] In some embodiments, the acidic alpha-glucosidase includes at least one substitution in a position or set of positions selected from 62 / 89 / 830, 62 / 830, 62 / 830 / 860, 62 / 830 / 860 / 932, and 62 / 830 / 932, where these positions are numbered with reference to Sequence ID No. 3104. In some embodiments, the acidic alpha-glucosidase includes at least one substitution in a position or set of positions selected from 62A / 89D / 830Q, 62A / 830Q, 62A / 830Q / 860F, 62A / 830Q / 860F / 932S, where these positions are numbered with reference to Sequence ID No. 3104. In some embodiments, the acidic alpha-glucosidase includes at least one substitution in a position or set of positions selected from L62A / A89D / K830Q, L62A / K830Q, L62A / K830Q / L860F, L62A / K830Q / L860F / A932S, and L62A / K830Q / A932S, where these positions are numbered with reference to Sequence ID No. 3104.

[0021] In some embodiments, recombinant acid alpha-glucosidase contains at least one mutation at at least one position, as provided in Tables 3-1, 3-2, 4-1, 6-1, 10-1, 10-2, 12-1, 13-1, 13-2, 14-1, 14-2, 15-1, 16-1, 17-1, 17-2, 17-3, 17-4, 17-5, 17-6, 17-7, 17-8, and / or 17-9. In some additional embodiments, recombinant acid alpha-glucosidase is derived from human acid alpha-glucosidase. In some further additional embodiments, recombinant acid alpha-glucosidase contains the polypeptide sequence of SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104.

[0022] In some additional embodiments, the recombinant acid alpha-glucosidase provided herein is thermally stable. In some further embodiments, the recombinant acid alpha-glucosidase exhibits resistance to proteolysis. In some further embodiments, the recombinant acid alpha-glucosidase exhibits resistance to at least one gastrointestinal protease. In some embodiments, the gastrointestinal protease is selected from chymotrypsin, trypsin, carboxypeptidase, and elastase. In some further embodiments, the recombinant acid alpha-glucosidase is acid-stable. In some additional embodiments, the recombinant acid alpha-glucosidase is stable to acidic and neutral pH. In some further embodiments, the recombinant acid alpha-glucosidase is purified. In some further embodiments, recombinant acid alpha-glucosidase exhibits at least one improved property compared to the reference sequence, which is selected from any combination of i) enhanced catalytic activity, ii) increased expression, iii) increased stability at neutral pH levels, iv) increased stability at acidic pH levels, iv) enhanced activity in cell lysates, and vi) decreased immunogenicity, or any combination of i), ii), iii), iv), v), and / or vi). In some embodiments, the reference sequence is selected from SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104. In some further embodiments, the recombinant acid alpha-glucosidase is more stable at pH 7 than the acid alpha-glucosidases of SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104.Furthermore, in some other embodiments, recombinant acid alpha-glucosidase is more stable at pH 4 than the acid alpha-glucosidase of SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104. In some further embodiments, recombinant acid alpha-glucosidase shows increased expression compared to the acid alpha-glucosidase of SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104. In some additional embodiments, recombinant acid alpha-glucosidase is more lysosomal stable than the acid alpha-glucosidase of SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104. In some further embodiments, recombinant acid alpha-glucosidase is more readily taken up by cells than acid alpha-glucosidase of SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104. In some additional embodiments, recombinant acid alpha-glucosidase exhibits greater enzymatic activity in cell lysates than acid alpha-glucosidase of SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104. In some additional embodiments, recombinant acid alpha-glucosidase exhibits reduced or decreased immunogenicity compared to acid alpha-glucosidase of SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104. In some embodiments, recombinant acid alpha-glucosidase is purified.

[0023] In some further embodiments, the recombinant acid alpha-glucosidase comprises a polypeptide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with at least one of the even-numbered sequences from SEQ ID NOs: 8 to 3378. In some embodiments, the recombinant acid alpha-glucosidase comprises a polypeptide sequence that is at least 90% identical to at least one of the even-numbered sequences from SEQ ID NOs: 8 to 3378. In some further embodiments, the recombinant acid alpha-glucosidase comprises a polypeptide sequence that includes at least one of the even-numbered sequences from SEQ ID NOs: 2 to 3378. In some further embodiments, the recombinant acid alpha-glucosidase comprises a polypeptide sequence that includes at least one of the even-numbered sequences from SEQ ID NOs: 8 to 3378.

[0024] The present invention also provides compositions comprising at least one recombinant acid alpha-glucosidase provided herein. In some embodiments, the composition comprises one recombinant acid alpha-glucosidase provided herein.

[0025] The present invention also provides recombinant polynucleotide sequences encoding at least one recombinant acid alpha-glucosidase provided herein. In some embodiments, the recombinant polynucleotide sequence encodes one recombinant acid alpha-glucosidase. In some embodiments, the recombinant polynucleotide sequence is selected from DNA, RNA, and mRNA. In some embodiments, the polynucleotide sequence is codon-optimized. In some further embodiments, the recombinant polynucleotide sequence encodes a recombinant acid alpha-glucosidase comprising a polypeptide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with respect to at least one of the even-numbered sequences among SEQ ID NOs: 8-3378. In some further embodiments, the recombinant acid alpha-glucosidase encoded by a polynucleotide sequence comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to at least one of the even-numbered sequences among SEQ ID NOs: 8-3378.

[0026] In some further embodiments, the recombinant polynucleotide sequence is sequence-identical to at least one of the odd-numbered sequences from SEQ ID NOs. 7-3377 by at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99%. In some further embodiments, the recombinant polynucleotide sequence is sequence-identical to at least one of the odd-numbered sequences from SEQ ID NOs. 7-3377 by at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some additional embodiments, the recombinant polynucleotide sequence includes a sequence having at least 90% sequence identity to at least one of the odd-numbered sequences from SEQ ID NOs. 7-3377. In some further embodiments, the recombinant polynucleotide sequence includes odd-numbered sequences from sequence numbers 7 to 3377.

[0027] The present invention also provides an expression vector comprising a recombinant polynucleotide sequence encoding recombinant acid alpha-glucosidase. In some embodiments, the recombinant polynucleotide sequence is operably ligated to a control sequence. In some further embodiments, the control sequence is a promoter. In some additional embodiments, the promoter is a heterologous promoter. The present invention also provides an expression vector referred to herein as pDH. In some embodiments, the pDH vector comprises at least one polynucleotide sequence encoding acid alpha-glucosidase. In some additional embodiments, the pDH vector comprises at least one polynucleotide sequence encoding acid alpha-glucosidase provided herein. In some further embodiments, the pDH vector comprises at least one polynucleotide sequence selected from odd-numbered sequences among SEQ ID NOs: 1 to 3377. In some additional embodiments, the pDH vector comprises at least one polynucleotide sequence encoding acid alpha-glucosidase selected from even-numbered sequences among SEQ ID NOs: 2 to 3378. In some embodiments, the pDH vector comprises SEQ ID NOs: 3379, while in some other embodiments, the pDH vector comprises SEQ ID NOs: 3380. In some additional embodiments, SEQ ID NO: 1 in SEQ ID NO: 3379 is replaced with a different polynucleotide sequence. In some embodiments, SEQ ID NO: 1 in SEQ ID NO: 3379 is replaced with at least one polynucleotide sequence selected from odd-numbered sequences among SEQ ID NOs: 1-3377. In some additional embodiments, the pDH vector includes a plasmid provided in map in Figure 9, while in some other embodiments, the pDH vector includes a plasmid provided in map in Figure 10. In some embodiments, the “stuffer sequence” (i.e., the bla sequence) in the plasmid shown in Figure 10 is replaced with the gene of interest. In some embodiments, the “stuffer gene” is numbered from the ATG start codon to the last codon of the gene from base pairs 724 to 1581 (for a total of 858 base pairs). As used herein, the terms “stuffer gene” and “stuffer sequence” refer to the sequence in the plasmid vector that is replaced with the gene of interest.As used herein, the term “target gene” refers to the gene encoding the desired polypeptide (e.g., “target polypeptide”). In some additional embodiments, the stuffer sequence is replaced with the target gene (i.e., the gene whose expression is desired in order to produce the target polypeptide, such as variant acid alpha-glucosidase).

[0028] The present invention also provides host cells comprising at least one expression vector provided herein. In some embodiments, the expression vector provided within the host cell is pDH. In some embodiments, the host cell is selected from eukaryotes and prokaryotes. In some further embodiments, the host cell is a mammalian cell.

[0029] The present invention also provides a method for producing recombinant acid alpha-glucosidase variants, comprising the step of culturing at least one host cell provided herein under conditions that produce acid alpha-glucosidase encoded by a recombinant polynucleotide. In some embodiments, the method further comprises the step of recovering the acid alpha-glucosidase. In some further embodiments, the method further comprises the step of purifying the acid alpha-glucosidase. The present invention also provides recombinant acid alpha-glucosidase variants produced according to the methods provided herein.

[0030] The present invention also provides compositions comprising at least one recombinant acid alpha-glucosidase provided herein. The present invention also provides for the use of the compositions provided herein. In some embodiments, the present invention provides a pharmaceutical composition for the treatment of Pompe disease comprising at least one composition provided herein. In some additional embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier and / or excipient. In some further embodiments, the pharmaceutical composition is suitable for parenteral injection or infusion into humans. In some further additional embodiments, the present invention provides a pharmaceutical composition comprising at least one recombinant polynucleotide provided herein. In some further further embodiments, the present invention provides a pharmaceutical composition comprising at least one recombinant polypeptide provided herein. In some further additional embodiments, the present invention provides a composition comprising at least one recombinant polynucleotide and at least one recombinant polypeptide provided herein.

[0031] The present invention also provides a method for treating and / or preventing symptoms of Pompe disease in a subject, comprising the steps of preparing a subject having Pompe disease and at least one pharmaceutical composition provided herein, and administering the pharmaceutical composition to the subject. In some embodiments, the symptoms of Pompe disease are improved. In some additional embodiments, the subject is an infant or a child. In some further embodiments, the subject is an adult or a young adult. [Brief explanation of the drawing]

[0032] [Figure 1] Figure 1 provides a graph showing the kinetic assay results for the three GAA variants described in Example 9.

[0033] [Figure 2] Figure 2 provides a graph showing the glycogen hydrolysis activity to glucose of the four GAA variants described in Example 9.

[0034] [Figure 3] Figure 3 provides a graph showing the 4-MuGlu hydrolysis levels of the four GAA variants described in Example 9.

[0035] [Figure 4] Figure 4 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after 5 hours of incubation at 37°C with cultured Pompe patient fibroblasts, followed by 24 hours of incubation.

[0036] [Figure 5] Figure 5 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after 24 hours of incubation at 37°C with cultured Pompe patient fibroblasts.

[0037] [Figure 6] Figure 6 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after 5 hours of incubation at 37°C with cultured C2C12 GAA- / - myoblasts, followed by 24 hours of incubation.

[0038] [Figure 7] Figure 7 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after 24 hours of incubation at 37°C with cultured C2C12 GAA- / - myoblasts.

[0039] [Figure 8] Figure 8 provides a graph showing the cellular uptake of different purified GAA variants, expressed as relative activity, after incubation at 37°C for 1–7 days with cultured Pompe patient fibroblasts.

[0040] [Figure 9] Figure 9 provides a plasmid map of pDH vectors containing WT GAA.

[0041] [Figure 10] Figure 10 provides a plasmid map showing a pDH vector containing the staffer sequence bla (i.e., beta-lactamase).

[0042] [Figure 11] Figure 11 provides a graph showing the stability of eight GAA variants at neutral pH and 37°C, expressed as normalized residual RFU activity.

[0043] [Figure 12] Figure 12 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after 24 hours of incubation at 37°C with cultured Pompe patient fibroblasts.

[0044] [Figure 13] Figure 13 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after 72 hours of incubation at 37°C with cultured Pompe patient fibroblasts.

[0045] [Figure 14] Figure 14 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after 24 hours of incubation at 37°C with cultured C2C12 GAA- / - myoblasts.

[0046] [Figure 15] Figure 15 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after incubation at 37°C for 72 hours with cultured C2C12 GAA- / - myoblasts.

[0047] [Figure 16]Figure 16 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after incubation at 37°C for 4 hours with cultured Pompe patient fibroblasts, followed by cell washing and a further 20-hour incubation.

[0048] [Figure 17] Figure 17 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after 24 hours of incubation at 37°C with cultured Pompe patient fibroblasts.

[0049] [Figure 18] Figure 18 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after 72 hours of incubation at 37°C with cultured Pompe patient fibroblasts.

[0050] [Figure 19] Figure 19 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after incubation at 37°C for 4 hours with cultured C2C12 GAA- / - myoblasts, followed by cell washing and a further 20-hour incubation.

[0051] [Figure 20] Figure 20 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after 24 hours of incubation at 37°C with cultured C2C12 GAA- / - myoblasts.

[0052] [Figure 21] Figure 21 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after 72 hours of incubation at 37°C with cultured C2C12 GAA- / - myoblasts.

[0053] [Figure 22]Figure 22 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after 96 hours of incubation at 37°C with cultured Pompe patient fibroblasts.

[0054] [Figure 23] Figure 23 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after 96 hours of incubation at 37°C with cultured C2C12 GAA- / - myoblasts.

[0055] [Figure 24] Figure 24 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after 48 hours of incubation at 37°C with cultured Pompe patient fibroblasts.

[0056] [Figure 25] Figure 25 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after incubation at 37°C for 48 hours with cultured C2C12 GAA- / - myoblasts.

[0057] [Figure 26] Figure 26 provides a graph showing the cellular uptake of different purified GAA variants, expressed as RFU activity, after incubation at 37°C for 1 hour with cultured Pompe patient fibroblasts, followed by cell washing and a further 71-hour incubation.

[0058] [Figure 27] Figure 27 provides a graph showing the cell uptake of different purified GAA variants, expressed as RFU activity, after incubation at 37°C for 1 hour with cultured C2C12 GAA- / - myoblasts, followed by cell washing and a further 71-hour incubation. [Modes for carrying out the invention]

[0059] Description of the Invention This invention provides engineered acid alpha-glucosidase (GAA) polypeptides and compositions thereof. In some embodiments, the engineered GAA polypeptides are optimized to provide enhanced catalytic activity and enhanced acid stability while reducing sensitivity to proteolysis. This invention also provides methods for utilizing compositions comprising engineered GAA polypeptides for therapeutic and other purposes. Abbreviations and definitions:

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. In general, the nomenclature used herein, as well as the experimental procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry, and nucleic acid chemistry described below, are well known and commonly used in the art. Such techniques are well known and described in a great many textbooks and references well known to those skilled in the art. Standard techniques or modified forms thereof are used in chemical synthesis and chemical analysis. All patents, patent applications, papers, and publications referred to herein both above and below are thus expressly incorporated herein by reference.

[0061] Any preferred methods and materials similar to or equivalent to those described herein may be used in carrying out the present invention, although some methods and materials are described herein. It should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, for these methodologies, protocols, and reagents may vary depending on the circumstances in which they are used by those skilled in the art. Therefore, the terms defined immediately below are better explained by referring to this application as a whole. All patents, patent applications, papers, and published documents referred to both above and below herein are thus explicitly incorporated herein by reference.

[0062] Furthermore, as used herein, the singular forms "a," "an," and "the" include plural references unless otherwise explicitly indicated by the context.

[0063] Numerical ranges include the number that defines the range. Therefore, any numerical range disclosed herein is intended to encompass all narrower numerical ranges that fall within such a wider range, as if all such narrower ranges were explicitly stated herein. Similarly, any maximum (or minimum) numerical limit disclosed herein is intended to encompass all numerical lower (or upper) limits, as if those lower (or upper) limits were explicitly stated herein.

[0064] The term "approximately" refers to the acceptable error for a particular value. In some examples, "approximately" means within 0.05%, 0.5%, 1.0%, or 2.0% of a given value range. In some examples, "approximately" means within 1, 2, 3, or 4 standard deviations of a given value. In some examples, "approximately" encompasses values that are 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.

[0065] Furthermore, the headings provided herein are not intended to limit the various aspects or embodiments of the invention that may be understood by referring to the application as a whole. Therefore, the terms defined immediately below are more fully defined by referring to the application as a whole. Nevertheless, for the sake of facilitating understanding of the invention, some terms are defined below.

[0066] Unless otherwise specified, nucleic acids are written from left to right in the 5' to 3' direction, and amino acid sequences are written from left to right in the amino to carboxy direction.

[0067] As used herein, the terms “comprising” and their cognates are used in their inclusive sense (i.e., equivalent to “including” and their corresponding cognates).

[0068] 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 numerical classification system for enzymes based on the chemical reactions they catalyze.

[0069] "ATCC" refers to the American Type Culture Collection, whose biorepository collection includes genes and cell lines.

[0070] "NCBI" refers to the National Center for Biological Information (NCBI) and the sequence databases it provides.

[0071] As used herein, the terms “acidic alpha-glucosidase,” “acidic α-glucosidase,” “acidic alpha-glucosidase polypeptide,” “lysosomal alpha-glucosidase,” and “GAA” refer to enzymes within a family of enzymes that break down glycogen present in lysosomes (EC 3.2.1.20). These enzymes may also be called “alpha-1,4-glucosidase,” “α-1,4-glucosidase,” “acidic maltase,” “glucoinvertase,” “glucosidosucrase,” “lysosomal alpha-glucosidase,” “lysosomal α-glucosidase,” “maltase,” or “maltase-glucoamylase.” One reaction catalyzed by the enzyme is the hydrolysis of a terminal, non-reducing (1→4) linked alpha-D-glucose residue, which results in the release of alpha-D-glucose.

[0072] As used herein, “Pompe disease” refers to type II glycogen storage disorder, an autosomal recessive genetic disorder resulting in a metabolic disorder characterized by lysosomal storage of glycogen in skeletal muscle and other tissues. Pompe disease is characterized based on age of onset, organ involvement, severity, and rate of progression. The more severe form is infant-onset Pompe disease (IOPD), which occurs in infancy. Another form, called “late-onset Pompe disease” (LOPD), occurs in individuals whose disease develops before 12 months of age but without IOPD-related cardiomyopathy, and in all individuals whose disease develops after 12 months of age. Synonyms for Pompe disease include “acid alpha-glucosidase deficiency,” “acid maltase deficiency,” “GAA deficiency,” “type II glycogen storage disorder,” “GSD II,” “GSD2,” and “type II glycogen storage disease.”

[0073] In this specification, "protein," "polypeptide," and "peptide" are used synonymously 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).

[0074] In this specification, "amino acids" are referred to by either their commonly known three-letter symbols or the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Nucleotides may similarly be referred to by their commonly accepted single-letter codes.

[0075] The terms “manipulated,” “recombinant,” “not naturally occurring,” and “variant,” when used in reference to cells, polynucleotides, or polypeptides, refer to a material or a material corresponding to the natural or native form of that material, which has been modified in a manner not normally occurring in nature, or which is identical but produced or derived from a synthetic material and / or through operations using recombinant techniques.

[0076] As used herein, “wild-type” and “naturally occurring” refer to forms found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence that is present in living organisms, can be isolated from a natural source, and has not been intentionally modified by human intervention.

[0077] The "coding sequence" refers to the portion of nucleic acid (for example, a gene) that codes for the amino acid sequence of a protein.

[0078] The term “sequence identity percentage (%)” is used herein to refer to a comparison between polynucleotides and polypeptides, determined by comparing two optimally aligned sequences across a comparison window, where portions of the polynucleotide or polypeptide sequences within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions in which identical nucleic acid bases or amino acid residues appear in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Alternatively, the percentage can be calculated by determining the number of positions in which identical nucleic acid bases or amino acid residues appear in both sequences or the number of positions in which nucleic acid bases or amino acid residues are aligned using gaps, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. It will be understood by those skilled in the art that there are numerous established algorithms available for the alignment of two sequences. The 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. 2:482

[1981] ), the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443

[1970] ), the similarity search method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444

[1988] ), computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection.Examples of algorithms suitable for determining sequence identity percentages and sequence similarity percentages include, but are not limited to, the BLAST and BLAST 2.0 algorithms described by Altschul et al. (see Altschul et al., J. Mol. Biol., 215: 403-410

[1990] ; and Altschul et al., 1977, Nucleic Acids Res., 3389-3402

[1977] , respectively). Software for performing BLAST analysis is publicly available through the website of the National Center for Biotechnology Information. This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive threshold score T when aligned with words of the same length in the database sequence. T is called the adjacent word score threshold (see Altschul et al., cited above). These initial adjacent word hits serve as seeds to initiate a search for longer HSPs containing them. Thus, word hits are expanded in both directions along each sequence as long as the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matched residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a score matrix is used to calculate the cumulative score. The expansion of word hits in each direction stops when the cumulative alignment score falls by X amounts from its maximum attainment; stops when the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments; or stops when the end of either sequence is reached. 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 a comparison of both strands. For amino acid sequences, the BLASTP program uses, by default, a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 score matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

[1989] ). Exemplary determination of sequence alignment and sequence identity % can be performed using the BESTFIT or GAP programs of the GCG Wisconsin Software package (Accelrys, Madison WI), using the provided default parameters.

[0079] As used herein, the term “reference sequence” refers to a defined sequence used as a criterion for sequence comparison. A reference sequence may also be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is a nucleic acid or polypeptide of at least 20 nucleotides or amino acid residues, at least 25 residues, at least 50 residues, at least 100 residues, or full length. 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 “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, the “reference sequence” may be based on a primary amino acid sequence, in which case the reference sequence is a sequence that may have one or more variations of the primary sequence. A "comparison window" refers to a conceptual segment of at least approximately 20 consecutive nucleotide positions or amino acid residues that allows a given sequence to be compared to a reference sequence of at least 20 consecutive nucleotides or amino acids, where the portion of the sequence within the comparison window may contain 20 percent or less of additions or deletions (i.e., gaps) compared to the reference sequence (which contains neither additions nor deletions) for optimal alignment of the two sequences. A comparison window may be longer than 20 consecutive residues and may include windows of 30, 40, 50, 100 or longer, as needed.

[0080] When used in the context of numbering a given amino acid or polynucleotide sequence, "correspond to," "reference to," or "compared to" queries the numbering of residues in a specified reference sequence when the given amino acid or polynucleotide sequence is compared to a reference sequence. In other words, the residue numbers or residue positions of a given polymer are indicated relative to the reference sequence, rather than by the actual number positions of the residues in the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, e.g., the amino acid sequence of an engineered GAA, can be aligned with a reference sequence by introducing gaps to optimize 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 relative to the reference sequence to which it is aligned.

[0081] An "amino acid difference" or "residue difference" refers to the difference between an amino acid residue at a given position in a polypeptide sequence and an amino acid residue at a corresponding position in a 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 that serves as the basis for the residue difference. For example, "residue difference at position X27 compared to SEQ ID NO: 2" refers to the difference in the amino acid residue at the polypeptide position corresponding to position 27 of SEQ ID NO: 2. Therefore, if the reference polypeptide of SEQ ID NO: 2 has phenylalanine at position 2, then "residue difference at position X27 compared to SEQ ID NO: 2" refers to the amino acid substitution of any residue other than phenylalanine at the polypeptide position corresponding to position 27 of SEQ ID NO: 2. In most examples herein, a specific amino acid residue difference at a given position is indicated as "XnY," where "Xn" specifies 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 (as shown, for example, in Tables 3-1, 3-2, 4-1, 6-1, 10-1, 10-2, 12-1, 13-1, 13-2, 14-1, 14-2, 15-1, 16-1, 17-1, 17-2, 17-3, 17-4, 17-5, 17-6, 17-7, 17-8, and / or 17-9), the Disclosure also provides specific amino acid differences, indicated by the conventional notation “AnB,” where A is a one-letter identifier of a residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is a one-letter identifier of a residue substitution in the sequence of the manipulated polypeptide. In some cases, the polypeptides of the Disclosure may contain one or more amino acid residue differences compared to a reference sequence, and these differences are indicated by a list of specified positions where the residue differences exist compared to the reference sequence. In some embodiments, where more than one amino acid may be used at the designated residue position of the polypeptide, the various amino acid residues that may be used are separated by " / " (e.g., X27P / X27R or X27P / R). In some embodiments, the enzyme variant contains more than one substitution.These substitutions are separated by slashes for readability (e.g., F27P / C944W). In some cases, the "X" does not precede the position number in this application. This application includes an engineered polypeptide sequence comprising one or more amino acid differences, which include either / or both conserved and non-conserved amino acid substitutions.

[0082] "Conservative amino acid substitution" refers to the substitution of a residue at a different residue having a similar side chain, and therefore usually includes the substitution of an amino acid in a polypeptide with an amino acid within the same or similar defined class of amino acids. For example, but not limited to, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain may be substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain may be substituted with another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain may be substituted with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain may be substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid may be replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0083] A “non-conservative substitution” refers to the substitution of an amino acid in a polypeptide with an amino acid that has significantly different side-chain properties. Non-conservative substitutions may use amino acids between defined groups rather than within the defined group and affect (a) the structure of the peptide backbone within the region of the substitution (e.g., substitution of glycine with proline), (b) charge or hydrophobicity, or (c) side-chain bulk. For example, exemplary non-conservative substitutions, not limited to, may include acidic amino acids substituted with basic or aliphatic amino acids, aromatic amino acids substituted with smaller amino acids, and hydrophilic amino acids substituted with hydrophobic amino acids.

[0084] "Deletion" refers to the modification of a polypeptide by the removal of 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, while retaining the enzymatic activity of the manipulated enzyme and / or improved properties. Deletions may relate to the internal and / or terminal portions of the polypeptide. In various embodiments, deletions may include contiguous segments or be discontinuous.

[0085] "Insertion" refers to the modification of a polypeptide by the addition of one or more amino acids from a reference polypeptide. Insertions may be located within the interior of the polypeptide or at the carboxyl or amino terminus. As used herein, insertions include fusion proteins known in the art. Insertions may be a continuous segment of amino acids in a naturally occurring polypeptide, or they may be separated by one or more amino acids.

[0086] As used synonymously in this specification, “functional fragment” or “bioactive fragment” refers to a polypeptide having an amino-terminal and / or carboxy-terminal deletion and / or an internal deletion, but whose remaining amino acid sequence is identical to the corresponding position in the sequence it is being compared to (e.g., the fully-length manipulated GAA of the present invention), and which retains substantially all of the activity of the full-length polypeptide.

[0087] "Isolated polypeptide" refers to a polypeptide that has been substantially separated from other naturally associated contaminants (e.g., proteins, lipids, and polynucleotides). This term encompasses polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., host cells or in vitro synthesis). Recombinant GAA polypeptides may be present in cells, in cell culture media, or prepared in various forms such as lysates or isolated preparations. Therefore, in some embodiments, recombinant GAA polypeptides may be isolated polypeptides.

[0088] A “substantially pure polypeptide” refers to a composition in which the polypeptide species is the dominant species present (i.e., its abundance in the composition is greater than any other individual polymer species, on a molar or weight basis), and is generally considered substantially purified if the species of interest accounts for at least about 50 percent of the molar or weight percent of the polymer species present. Generally, a substantially pure GAA composition consists of 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, in terms of molar or weight percent of all polymer species present in the composition. In some embodiments, the species of interest is purified to an intrinsically homogeneous state in which the composition is essentially a single polymer species (i.e., the contaminants cannot be detected in the composition by conventional detection methods). Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered polymer species. In some embodiments, an isolated recombinant GAA polypeptide is a substantially pure polypeptide composition.

[0089] "Improved enzymatic properties" refers to an engineered GAA polypeptide that exhibits improvements in any enzymatic properties compared to a reference GAA polypeptide and / or similar to a wild-type GAA polypeptide or another engineered GAA polypeptide. Improved properties include, but are not limited to, properties such as increased protein expression, increased thermal activity, increased thermal stability, increased pH activity, increased stability, increased enzyme activity, increased substrate specificity or affinity, increased specific activity, increased resistance to substrate or end-product inhibition, increased chemical stability, improved chemoselectivity, improved solvent stability, increased tolerance to acidic, neutral or basic pH, increased resistance 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.

[0090] "Increased enzyme activity" or "enhanced enzyme activity" refers to improved properties of an engineered GAA polypeptide, which may be expressed by an increase in specific activity (e.g., product produced / time / protein weight) or an increase in the percentage of substrate conversion to product (e.g., the percentage of substrate conversion to product within a specified period using a specified amount of GAA) compared to a reference GAA enzyme. Exemplary methods for determining enzyme activity are provided in the examples. Any property related to enzyme activity may be affected, and such properties may include K, whose change may lead to an increase in enzyme activity. m , V max or k cat The classic enzymatic properties are included. The improvement in enzymatic activity can range from approximately 1.1 times the enzymatic activity of the corresponding wild-type enzyme to as much as 2, 5, 10, 20, 25, 50, 75, 100, 150, 200 times, or even higher, the enzymatic activity of the GAA polypeptide derived from the naturally occurring GAA or another engineered GAA.

[0091] In some embodiments, the manipulated GAA polypeptide is transported at a rate of at least 0.1 / second, at least 0.5 / second, at least 1.0 / second, at least 5.0 / second, at least 10.0 / second, and in some preferred embodiments, more than 10.0 / second, k cat It has K m The concentrations are in the range of approximately 1 μM to approximately 5 mM, approximately 5 μM to approximately 10 mM, approximately 30 μM to approximately 30 mM, or approximately 50 μM to approximately 50 mM. In some specific embodiments, the manipulated GAA enzyme exhibits an improvement in enzyme activity of 1.5 to 10 times, 1.5 to 25 times, 1.5 to 50 times, 1.5 to 100 times, or greater, of that of a reference GAA enzyme (e.g., wild-type GAA or any other reference GAA, e.g., SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104) after exposure to certain conditions.

[0092] GAA 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 high-performance liquid chromatography (HPLC) separation combined with UV absorbance or fluorescence detection. In some embodiments, the amount of product produced can be measured by monitoring fluorescence (Ex. 355 nm, Em. 460 nm) after hydrolysis of the 4-methylumbelliferyl-alpha-D-glucopyranoside (4-MUGlu) molecule. Comparison of enzyme activity is performed using defined preparations of the enzyme, defined assays under set conditions, and one or more defined substrates, as further described in detail herein. Generally, when lysates are compared, the number of cells and the amount of protein to be assayed are determined, and the use of identical expression systems and identical host cells is also determined to minimize variations in the amount of enzyme produced by host cells and present in the lysates.

[0093] The term "improved tolerance to acidic pH" means that the recombinant GAA according to the present invention will have improved stability (higher retained activity at approximately pH 4.8 after exposure to acidic pH for a specified period (e.g., 1 hour, up to 24 hours)) compared to a reference GAA or another enzyme.

[0094] The term "improved tolerance to neutral pH" means that the recombinant GAA according to the present invention will have improved stability (higher retained activity at approximately pH 7 after exposure to neutral pH for a specified period (e.g., 1 hour, up to 24 hours)) compared to a reference GAA or another enzyme.

[0095] The term “improved cell uptake” means that the recombinant GAA provided herein exhibits increased endocytosis into cells compared to a reference GAA (including wild-type GAA) or another enzyme. In some embodiments, the cells are cultured Pompe patient cells (which retained a higher degree of intracellular activity after incubation of cultured cells over a specified period compared to a reference GAA or another enzyme). In some additional embodiments, the recombinant GAA provided herein exhibits greater intracellular activity retained in cultured cells over a specified period compared to a reference GAA (including wild-type GAA) or another enzyme. In some additional embodiments, the period is approximately 4 hours, while 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 longer than 4 hours (e.g., 5, 6, 7, 8, or longer).

[0096] The terms “reduced immunogenicity” and “decreased immunogenicity” mean that the recombinant GAAs provided herein induce a reduced immune response compared to wild-type or another reference GAA.

[0097] As used herein, "physiological pH" means the pH range commonly found in the blood of the subject (e.g., human).

[0098] The term "basic pH" (used, for example, in relation to improved stability to basic pH conditions or increased tolerance to basic pH) refers to a pH range of approximately 7 to 11.

[0099] The term "acidic pH" (used, for example, in relation to improved stability to acidic pH conditions or increased tolerance to acidic pH) refers to a pH range of approximately 1.5 to 4.5.

[0100] "Conversion" refers to the enzymatic (or in vivo) conversion of a substrate to its corresponding product. "Conversion percentage" refers to the percentage of a substrate converted to the product within a specified period under specified conditions. Therefore, the "enzymatic activity" or "activity" of a GAA polypeptide can be expressed as the "conversion percentage" of the substrate to the product within a specific period.

[0101] "Hybridization stringency" refers to hybridization conditions in nucleic acid hybridization, such as washing conditions. Generally, hybridization reactions are carried out under lower stringency conditions, followed by washing with varying, but higher, stringency levels. The term "moderately stringent hybridization" refers to conditions that allow target DNA to bind to complementary nucleic acids having approximately 60% identity to the target DNA, preferably approximately 75% identity, approximately 85% identity, and higher than approximately 90% identity to the target polynucleotide. An exemplary moderately stringent condition corresponds to hybridization in 50% formamide, 5× Denhart solution, 5× SSPE, 0.2% SDS at 42°C, followed by washing in 0.2× SSPE, 0.2% SDS at 42°C. "Highly stringent hybridization" refers to conditions where the thermal fusion temperature T is determined for a defined polynucleotide sequence under solution conditions. mThis generally refers to conditions where the temperature is approximately 10°C or lower. In some embodiments, high-stringency conditions refer to conditions that allow hybridization of only nucleic acid sequences that form a stable hybrid at 65°C in 0.018M NaCl (i.e., if the hybrid is not stable at 65°C in 0.018M NaCl, it will not be stable under high-stringency conditions as intended herein). High-stringency conditions can be provided, for example, by hybridization under conditions equivalent to 50% formamide, 5× Denhart solution, 5× SSPE, and 0.2% SDS at 42°C, followed by washing at 65°C in 0.1× SSPE and 0.1% SDS. Another high-stringency condition is hybridization under conditions equivalent to hybridization at 65°C in 5× SSC containing 0.1% (w:v) SDS and washing at 65°C in 0.1× SSC containing 0.1% SDS. Other highly stringent hybridization conditions, as well as moderately stringent conditions, are described in the references above.

[0102] "Codon-optimized" refers to the modification of codons in a protein-coding polynucleotide to codons preferred in a particular organism, so that the encoded protein is expressed more efficiently in that organism. While the genetic code is degenerate, with most amino acids represented by a few codons called "synonyms" or "synonymous" codons, it is well known that codon usage by a particular organism is not random but biased towards specific codon triplets. This bias in codon usage can be more pronounced with respect to a given gene, a gene of common function or ancestral origin, a protein highly expressed for low-copy-number proteins, and the aggregated protein-coding regions of an organism's genome. In some embodiments, a polynucleotide encoding a GAA enzyme may be codon-optimized for optimal production from a host organism selected for expression.

[0103] In this specification, "control sequence" refers to all components necessary or advantageous for the expression of the polynucleotide and / or polypeptide of the Application. Each control sequence may be native or heterogeneous to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter sequence, signal peptide sequence, start sequence, and transcription terminator. At a minimum, a control sequence includes a promoter, as well as transcription and translation stop signals. A control sequence may also include a linker to introduce a specific restriction site that facilitates ligation between the control sequence and the coding region of the nucleic acid sequence encoding the polypeptide.

[0104] "Operationally linked" is defined herein as a configuration in which the control sequence is appropriately positioned relative to the polynucleotide of interest (i.e., in a functional relationship) to direct or regulate the expression of the polynucleotide and / or polypeptide of interest.

[0105] A "promoter sequence" refers to a nucleic acid sequence, such as a coding sequence, that is recognized by a host cell for the expression of a polynucleotide of interest. The promoter sequence contains transcriptional regulatory sequences that mediate the expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence exhibiting transcriptional activity in a selected host cell, including mutant, cleavage, and hybrid promoters, and the promoter can be obtained from a gene encoding an extracellular or intracellular polypeptide that is either homogeneous or heterogeneous to the host cell.

[0106] "Preferred reaction conditions" refers to the conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme load, substrate load, temperature, pH, buffer, cosolvent, etc.) that enable the GAA polypeptide of this application to convert a substrate into a desired product compound. Exemplary "preferred reaction conditions" are provided herein and illustrated by the examples. "Load," as in "compound load" or "enzyme load," refers to the concentration or amount of a component 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 that is acted upon by the GAA polypeptide. "Product" in the context of an enzymatic conversion process refers to the compound or molecule that results from the action of the GAA polypeptide on the substrate.

[0107] As used herein, the term “culturing” refers to growing a population of microbial cells under any preferred conditions (e.g., using a liquid, gel, or solid medium).

[0108] Recombinant polypeptides can be produced using any suitable method known in the art. The gene encoding the desired wild-type polypeptide can be cloned into a vector such as a plasmid and expressed in a desired host such as E. coli or S. cerevisiae. Variants of recombinant polypeptides can be generated by various methods known in the art. Indeed, there are many different mutagenesis techniques well known to those skilled in the art. In addition, mutagenesis kits are available from many commercial molecular biology suppliers. Methods can be used to perform specific substitutions at defined amino acids (site-directed mutagenesis), specific or random mutations at local regions of a gene (site-directed mutagenesis), or random mutagenesis across an entire gene (e.g., saturation mutagenesis). A great many suitable methods for generating enzyme variants are known to those skilled in the art, including, but are not limited to, site-directed mutagenesis of single-stranded or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation sudden displacement, or any other suitable method known in the art. Non-limiting examples of methods used in DNA and protein engineering are provided in the following patents: U.S. Patent Nos. 6,117,679, 6,420,175, 6,376,246, 6,586,182, 7,747,391, 7,747,393, 7,783,428, and 8,383,346. After producing variants, they can be screened for any desired properties (e.g., high or increased activity, or low or decreased activity, increased thermal activity, increased thermal stability, and / or acidic pH stability). In some embodiments, “recombinant GAA polypeptides” (also referred herein as “engineered GAA polypeptides,” “variant GAA enzymes,” and “GAA variants”) are used.

[0109] As used herein, “vector” is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, the vector is an expression vector operably ligated to a preferred regulatory sequence capable of performing expression of a polypeptide encoded in the DNA sequence in a preferred host. In some embodiments, the “expression vector” has a promoter sequence operably ligated to a DNA sequence (e.g., an introduced gene) for driving expression in a host cell, and in some embodiments, also includes a transcriptional terminator sequence. In some preferred embodiments, the pDH vectors provided herein are used.

[0110] As used herein, the term “gene therapy vector” refers to a vehicle or carrier suitable for the delivery of a polynucleotide sequence to a cell. In some embodiments, the vector encapsulates a gene (e.g., a therapeutic gene) or polynucleotide sequence for delivery to a cell or tissue. Such vectors include, but are not limited to, adenovirus (AV) vectors, adeno-associated virus (AAV) vectors, lentivirus (LV) vectors, and non-viral vectors, such as liposomes. Since any vehicle suitable for a given setting can be used, the invention is not intended to be limited to any particular gene therapy vector. Gene therapy vectors can be designed to deliver a gene to a specific species or host, or they can find more general applicability.

[0111] As used herein, the term “expression” includes any steps involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses the secretion of polypeptides from cells.

[0112] As used herein, the term “produce” refers to the production of proteins and / or other compounds by a cell. This term is intended to encompass any steps involved in the production of polypeptides, 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.

[0113] As used herein, an amino acid or nucleotide sequence (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) is considered "heterogeneous" to another sequence with which it is operably linked if the two sequences do not naturally associate.

[0114] As used herein, the terms “host cell” and “host strain” refer to a suitable host for an expression vector containing DNA (e.g., a polynucleotide encoding a GAA variant) provided herein. In some embodiments, the host cell is a eukaryotic or prokaryotic cell into which a vector constructed using recombinant DNA techniques known in the art has been introduced or transfected by transformation.

[0115] The term “analog” refers to a polypeptide having sequence identity higher than 70% but less than 100% of a reference polypeptide (e.g., higher than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%). In some embodiments, the term analog refers to a polypeptide containing not only naturally occurring amino acids but also one or more non-naturally occurring amino acid residues, including but not limited to homoarginine, ornithine, and norvaline. In some embodiments, the analog also includes one or more D-amino acid residues and non-peptide linkages between two or more amino acid residues.

[0116] The term "therapeutic agent" refers to a compound administered to a subject exhibiting signs or symptoms of a pathological condition, which has a beneficial or desirable medical effect.

[0117] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in mammalian subjects (e.g., humans) and comprising a pharmaceutically effective amount of the manipulated GAA polypeptide encompassed by the present invention and an acceptable carrier.

[0118] The term “gene therapy” refers to the delivery of genes, polydeoxyribonucleotides, or polynucleotide sequences via gene therapy vectors to cells or tissues for the treatment or prevention of disease, or for the modification of those cells or tissues. Gene therapy may include replacing disease-causing mutant genes with healthy copies of those genes, or inactivating or “knocking out” improperly functioning mutant genes. In some embodiments, gene therapy is used to treat disease in patients.

[0119] The term "mRNA therapy" refers to the delivery of mRNA polyribonucleotide sequences to cells or tissues for the treatment or prevention of disease, or for the modification of those cells or tissues. In some embodiments, the mRNA polynucleotide sequences for delivery to cells or tissues are formulated, for example, but not limited to, liposomes. In some embodiments, mRNA therapy is used to treat disease in patients.

[0120] The term "cell therapy" refers to the delivery of exogenously modified living cells to a patient to supply missing genes for the treatment or prevention of disease. The modified cells are then reintroduced into the body.

[0121] The term "effective quantity" refers to the amount sufficient to produce the desired result. Those skilled in the art can determine what an effective quantity is by using routine experiments.

[0122] The terms “isolated” and “purified” are used to refer to molecules (e.g., isolated nucleic acids, polypeptides, etc.) or other components that have been removed from at least one other component from which they naturally associate. The term “purified” does not require absolute purity and is rather intended as a relative definition.

[0123] The term "subject" encompasses mammals, such as humans, non-human primates, livestock, companion animals, and laboratory animals (e.g., rodents and lagomorphs). This term is intended to include both males and females.

[0124] As used herein, the term “patient” means any subject who will be assessed for a disease, treated for a disease, or is experiencing a disease.

[0125] The term "infant" refers to a child from one month old to approximately one year old. As used herein, the term "neonatal" refers to a child from birth to 28 days old. The term "premature infant" refers to an infant born after 20 weeks of gestation but before full term, generally with a birth weight of approximately 500 to 2499 grams. A "very low birth weight infant" is an infant with a birth weight of less than 1500g.

[0126] As used herein, the term “child” refers to a person who has not reached the legal age to consent to a treatment or research procedure. In some embodiments, the term refers to a person between birth and adolescence.

[0127] As used herein, the term “adult” refers to a person who has reached the legal age of attainment in the relevant jurisdiction (e.g., 18 years in the United States). In some embodiments, the term refers to any fully grown, mature organism. In some embodiments, the term “young adult” refers to a person who is under 18 years of age but has reached sexual maturity.

[0128] As used herein, “composition” and “formulation” encompass products comprising at least one operated GAA of the present invention (e.g., pharmaceutical compositions, health / nutritional supplements, animal feed, etc.) for any preferred use.

[0129] The terms “administer” and “administer” the composition mean providing the composition of the present invention to a target (for example, a person affected by Pompe disease).

[0130] When used in relation to pharmaceutical compositions, the term "carrier" refers to any of the standard pharmaceutical carriers, buffers, and excipients, such as stabilizers, preservatives, and adjuvants.

[0131] The term "pharmaceutically acceptable" means a material that can be administered to a subject without causing any undesirable biological effects or harmfully interacting with any of the components it contains that have desirable biological activity.

[0132] As used herein, the term “excipient” refers to any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other component other than the active pharmaceutical ingredient (API; for example, the manipulated GAA polypeptide of the present invention). Excipients are typically included for formulation and / or administration.

[0133] The term "therapeutic dose," when used in relation to the symptoms of a disease / condition, refers to the amount and / or concentration of a compound (e.g., a modified GAA polypeptide) that improves, reduces or eliminates one or more symptoms of the disease / condition, or prevents or delays the onset of symptoms.

[0134] The term “therapeutic dose,” when used in relation to a disease / condition, refers to the amount and / or concentration of a composition (e.g., a modified GAA polypeptide) that improves, reduces, or eliminates the disease / condition. In some embodiments, the term is used in relation to the amount of a composition that elicits a biological (e.g., medical) response in a tissue, system, or animal subject, as sought by researchers, physicians, veterinarians, or other clinicians.

[0135] The terms “to treat,” “to treat,” and “treatment” are intended to encompass not only mitigating treatments but also preventive treatments (e.g., preventive treatments). Manipulated GAA polypeptide:

[0136] In some embodiments, the engineered GAA polypeptide is produced by culturing a microorganism containing at least one polynucleotide sequence encoding at least one engineered GAA polypeptide under conditions that promote the production of the engineered GAA polypeptide. In some embodiments, the engineered GAA polypeptide is recovered from the resulting culture medium and / or cells.

[0137] The present invention provides exemplary engineered GAA polypeptides having GAA activity. The examples provide a table showing sequence-structure information demonstrating the correlation between specific amino acid sequence features and the functional activity of the engineered GAA polypeptide. This structure-function correlation information is provided in the form of specific amino acid residue differences compared to a reference engineered polypeptide, as shown in the examples. The examples further provide experimentally determined activity data for the exemplary engineered GAA polypeptide. Polynucleotides encoding the manipulated polypeptide, expression vectors, and host cells:

[0138] The present invention provides polynucleotides encoding the engineered GAA polypeptide described herein. In some embodiments, the polynucleotides are operably ligated to one or more heterologous regulatory sequences that control gene expression to create recombinant polynucleotides capable of expressing the polypeptide. Expression constructs containing heterologous polynucleotides encoding the engineered GAA polypeptide can be introduced into suitable host cells to express the corresponding GAA polypeptide.

[0139] As will be apparent to those skilled in the art, knowledge of protein sequences and codons corresponding to various amino acids allows for a description of all polynucleotides that can encode a target polypeptide. The degeneracy of the genetic code, where the same amino acids are encoded by substitute or synonymous codons, makes it possible to create a very large number of nucleic acids, all of which encode the manipulated GAA 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 way that does not alter the amino acid sequence of the protein. In this regard, the present invention specifically intends for each and all possible variants of the polynucleotides encoding the polypeptides described herein, which can be made by selecting combinations based on possible codon options, and all such variants are considered to be specifically disclosed for all polypeptides described herein, including the variants provided in Tables 3-1, 3-2, 4-1, 6-1, 10-1, 10-2, 12-1, 13-1, 13-2, 14-1, 14-2, 15-1, 16-1, 17-1, 17-2, 17-3, 17-4, 17-5, 17-6, 17-7, 17-8 and / or 17-9, as well as SEQ ID NOs: 2, 8 and / or 14.

[0140] In various embodiments, codons are preferably selected to suit the host cell in which the protein will be produced. For example, preferred codons used in bacteria are used for expression in bacteria. Therefore, codon-optimized polynucleotides encoding the engineered GAA 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.

[0141] In some embodiments, as described above, the polynucleotide is an engineered polypeptide having GAA activity having the properties disclosed herein, with at least 80%, 85%, 86%, 87%, 88% relative to a reference sequence selected from SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880 and / or 3104. Amino acid sequences having 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity, or Table 3-1, 3-2, 4-1, 6-1, 10-1, 10-2, 12-1, 13-1, 13-2, 14-1, 14-2, 15-1, 16-1, 17-1, 17-2, 17-3, 17-4, 17-5, 17-6, 17- Encoding a polypeptide comprising the amino acid sequence of any variant as disclosed in 7, 17-8 and / or 17-9, and comprising one or more residue differences compared to the reference polypeptide of SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880 and / or 3104, or the amino acid sequence of any variant as disclosed in Tables 3-1, 3-2, 4-1, 6-1, 10-1, 10-2, 12-1, 13-1, 13-2, 14-1, 14-2, 15-1, 16-1, 17-1, 17-2, 17-3, 17-4, 17-5, 17-6, 17-7, 17-8 and / or 17-9 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue positions). In some embodiments, the reference sequence is selected from sequence numbers 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104.

[0142] The present invention provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with respect to SEQ ID NO: 2. The present invention provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 2. In some embodiments, acid alpha-glucosidase is 27, 27 / 944, 28, 29 / 478, 30, 88, 107, 109, 109 / 842, 110, 113, 135, 137, 138, 148, 150, 247, 274, 276, 278, 375, 403, 414, 418, 418 / 499, 421, 426, 437, 444, 455, 463, 471, The position or set of positions selected from 471 / 478, 476, 489, 527, 547, 581, 610, 642, 668, 670, 692, 725 / 732, 750, 753, 786, 820, 862, 871, 895, 897, 930, 934, and 944 includes at least one substitution, where these positions are numbered with reference to Sequence ID No. 2. In some embodiments, acid alpha-glucosidases include 27P, 27P / 944W, 27R, 28P, 28R, 28S, 29T / 478T, 30G, 30K, 30T, 88G, 88S, 107G, 107P, 109G / 842E, 109P, 110G, 110L, 113S, 135A, 135Q, 137P, 138A, 148G, 148Y, 150G, 247R, 274G, 276F, 276Y, 278A, 278G, 375E, 403W, 414P, 418E / 499R, 418R, 421S, 426R,It includes at least one substitution or set of substitutions at one or more positions selected from 437S, 444T, 455V, 463A, 471Q / 478S, 471S, 476A, 476H, 489R, 527R, 547G, 581G, 581T, 610A, 610G, 610S, 642M, 642Q, 642S, 668H, 670N, 692Q, 725N / 732I, 750P, 753T, 786P, 786Y, 820E, 862G, 871E, 895R, 897V, 930R, 934R, 944G, and 944R, where these positions are numbered with reference to Sequence ID No. 2. In some embodiments, the acidic alpha-glucosidases are F27P, F27P / C944W, F27R, L28P, L28R, L28S, L29T / A478T, V30G, V30K, V30T, K88G, K88S, Q107G, Q107P, L109G / G842E, L109P, Q110G, Q110L, Q11 3S, S135A, S135Q, E137P, M138A, T148G, T148Y, T150G, Q247R, D274G, A276F, A276Y, T2 78A, T278G, I375E, R403W, R414P, A418E / H499R, A418R, Q421S, G426R, A437S, A444T, R4 It includes at least one substitution or set of substitutions at one or more positions selected from 55V, E463A, K471Q / A478S, K471S, S476A, S476H, A489R, N527R, A547G, K581G, K581T, W610A, W610G, W610S, L642M, L642Q, L642S, S668H, L670N, T692Q, K725N / V732I, A750P, A753T, R786P, R786Y, G820E, R862G, L871E, K895R, T897V, C930R, L934R, C944G, and C944R, where these positions are numbered with reference to Sequence ID No. 2. In some embodiments, acid alpha-glucosidase is 29 / 218 / 240 / 668 / 700 / 744 / 869, 29 / 218 / 240 / 700 / 869, 29 / 240 / 596 / 668 / 700 / 744 / 869, 29 / 240 / 596 / 668 / 869, 36 / 106 / 150 / 218 / 527 / 750 / 883 / 894,106 / 112 / 150 / 218 / 414 / 527 / 793 / 883、106 / 150 / 169 / 218 / 414 / 486 / 527 / 750 / 894、106 / 150 / 169 / 218 / 414 / 486 / 527 / 894、106 / 150 / 169 / 218 / 414 / 486 / 749 / 793 / 883 / 894、106 / 150 / 169 / 218 / 414 / 486 / 750 / 793 / 883 / 894、106 / 150 / 169 / 218 / 414 / 486 / 793 / 883、106 / 150 / 169 / 218 / 414 / 486 / 894、106 / 150 / 169 / 218 / 414 / 749 / 750 / 793 / 883、106 / 150 / 169 / 218 / 414 / 749 / 793、106 / 150 / 169 / 218 / 414 / 749 / 793 / 883、106 / 150 / 169 / 218 / 486 / 527 / 749 / 793 / 894、106 / 150 / 169 / 218 / 486 / 749 / 883、106 / 150 / 169 / 218 / 486 / 883、106 / 150 / 169 / 218 / 749 / 800、106 / 150 / 169 / 414 / 486 / 749 / 750 / 883、106 / 150 / 169 / 527 / 749 / 793 / 883、106 / 150 / 169 / 749 / 793 / 883 / 894、106 / 150 / 218 / 331 / 414 / 486 / 527 / 733 / 749 / 793、106 / 150 / 218 / 414 / 486 / 642 / 750 / 793 / 883、106 / 150 / 218 / 414 / 486 / 750 / 793 / 894、106 / 150 / 218 / 414 / 527 / 749 / 750 / 883、106 / 150 / 218 / 414 / 527 / 749 / 793 / 883 / 894、106 / 150 / 218 / 414 / 749 / 750 / 793 / 883 / 894、106 / 150 / 218 / 414 / 749 / 793 / 883、106 / 150 / 218 / 486 / 527 / 749 / 894、106 / 150 / 218 / 486 / 793 / 883、106 / 150 / 218 / 527 / 749 / 750 / 793、106 / 150 / 218 / 527 / 793 / 894、106 / 150 / 218 / 749 / 750 / 793、106 / 150 / 218 / 793、106 / 150 / 218 / 793 / 894、106 / 150 / 245 / 793 / 883 / 894、106 / 150 / 414 / 749 / 750 / 793 / 894、106 / 150 / 414 / 749 / 793 / 894、106 / 150 / 486 / 527 / 750 / 793、106 / 150 / 486 / 749 / 793 / 883 / 894、106 / 150 / 749 / 793 / 883、106 / 169 / 185 / 218 / 414 / 749 / 750 / 793、106 / 191 / 280 / 402 / 414 / 444 / 727、106 / 191 / 414 / 444 / 522 / 928 / 944、106 / 191 / 414 / 489 / 928 / 944、106 / 280 / 402 / 414 / 444 / 489 / 727 / 944、150 / 169 / 218 / 414 / 527 / 793、150 / 218 / 414 / 486 / 749 / 750、150 / 218 / 414 / 486 / 750 / 793、150 / 218 / 414 / 486 / 750 / 793 / 883、150 / 218 / 414 / 749 / 750 / 793 / 894、150 / 218 / 414 / 749 / 793、150 / 218 / 527 / 749 / 793、150 / 218 / 749 / 750 / 793、150 / 218 / 749 / 793、150 / 414 / 486 / 527 / 750 / 894、150 / 414 / 486 / 749 / 750 / 793、150 / 486 / 750 / 883 / 894、169 / 486 / 750 / 793 / 883、180 / 275 / 402 / 518 / 547 / 610 / 638 / 669 / 671、180 / 402 / 431 / 507 / 547 / 610 / 669 / 671 / 793、180 / 402 / 507 / 547 / 610 / 671、191 / 280 / 402 / 414 / 444 / 465 / 842 / 928、191 / 280 / 402 / 414 / 444 / 489 / 500 / 944、191 / 280 / 414 / 444 / 489 / 500 / 522 / 842 / 928 / 944、191 / 280 / 414 / 444 / 489 / 522 / 727 / 944、191 / 280 / 414 / 489 / 842 / 928 / 944、191 / 280 / 414 / 944、191 / 414 / 522 / 842 / 944、196 / 402 / 431 / 547 / 610 / 638、218 / 668 / 700 / 869、224 / 402 / 507 / 518 / 547 / 638 / 668、269 / 275 / 431 / 518 / 547 / 638 / 668 / 669、275 / 281 / 402 / 431 / 507 / 518 / 610 / 668、275 / 281 / 402 / 431 / 518 / 547 / 610 / 669 / 671、275 / 281 / 402 / 507 / 518 / 547 / 638 / 669 / 671、275 / 281 / 402 / 518 / 547 / 610 / 638 / 671、275 / 281 / 402 / 518 / 547 / 610 / 668 / 669 / 887、275 / 281 / 402 / 547 / 610 / 638 / 669 / 671、275 / 281 / 431 / 518 / 547 / 638 / 669 / 671、275 / 281 / 507 / 547 / 669 / 671、275 / 281 / 610 / 638 / 668 / 669、275 / 281 / 671、275 / 377 / 402 / 507 / 518 / 669 / 671 / 715、275 / 402 / 431 / 507 / 547 / 671、275 / 402 / 431 / 518 / 610 / 638 / 669 / 671 / 922、275 / 402 / 507 / 547 / 610 / 638 / 668 / 669、275 / 402 / 507 / 547 / 610 / 638 / 669 / 671、275 / 402 / 507 / 547 / 610 / 671、275 / 402 / 547 / 610 / 638 / 669 / 671、275 / 402 / 547 / 638 / 669 / 671、275 / 402 / 638 / 669 / 671、275 / 431 / 507 / 518 / 547 / 668 / 669 / 671、275 / 431 / 507 / 518 / 610 / 669 / 671、275 / 431 / 507 / 547 / 610 / 638 / 671、275 / 431 / 518 / 547 / 638 / 668、275 / 431 / 518 / 610 / 638 / 669 / 671、275 / 431 / 638、275 / 507 / 518 / 547 / 610 / 638 / 668 / 669、275 / 507 / 518 / 547 / 638 / 669 / 671、275 / 507 / 547 / 610 / 638 / 669 / 671、275 / 507 / 547 / 668 / 669 / 671、275 / 518 / 671、280 / 402 / 536 / 928、281 / 402 / 507 / 518 / 547 / 610 / 638 / 669 / 671、281 / 402 / 507 / 547 / 638 / 669 / 671、281 / 402 / 518 / 547 / 610 / 638 / 668 / 669、281 / 402 / 518 / 547 / 668、281 / 431 / 507 / 518 / 547 / 610 / 638 / 668、402 / 431 / 518 / 547 / 610 / 668、402 / 431 / 518 / 547 / 671、402 / 431 / 518 / 610、402 / 431 / 547 / 638 / 671、The position or set of positions selected from 431 / 507 / 518 / 541 / 547 / 638 / 669 / 671, 431 / 507 / 518 / 669 / 671, 507 / 547 / 610, 507 / 547 / 638 / 669 / 671, 547 / 610 / 638 / 671, and 547 / 638 / 668 includes at least one substitution, The positions are numbered with reference to Sequence ID No. 2. In some embodiments, the acid alpha-glucosidases are 29Q / 218S / 240I / 668D / 700F / 744V / 869L, 29Q / 240I / 596P / 668D / 869L, 29Q / 240I / 596S / 668D / 700F / 744V / 869T, 29V / 218S / 240I / 700F / 869T, 36R / 106P / 150S / 218S / 527D / 750P / 883H / 894R, 106P / 112S / 150S / 218S / 414G / 527D / 793K / 883H, 106P / 150S / 169S / 218S / 414G / 486E / 527D / 750P / 894R, 106P / 150S / 169S / 218S / 414G / 486E / 527D / 894R, 106P / 150S / 169S / 218S / 414G / 486E / 749E / 793K / 883H / 894 R, 106P / 150S / 169S / 218S / 414G / 486E / 750P / 793K / 883H / 894R, 106P / 15 0S / 169S / 218S / 414G / 486E / 793K / 883H, 106P / 150S / 169S / 218S / 414G / 48 6E / 894R, 106P / 150S / 169S / 218S / 414G / 749E / 750P / 793K / 883H, 106P / 1 50S / 169S / 218S / 414G / 749E / 793K, 106P / 150S / 169S / 218S / 414G / 749E / 793K / 883H, 106P / 150S / 169S / 218S / 486E / 527D / 749E / 793K / 894R, 106P / 150S / 169S / 218S / 486E / 749E / 883H, 106P / 150S / 169S / 218S / 486E / 883 H, 106P / 150S / 169S / 218S / 749E / 800A, 106P / 150S / 169S / 414G / 486E / 74 9E / 750P / 883H, 106P / 150S / 169S / 527D / 749E / 793K / 883H, 106P / 150S / 1 69S / 749E / 793K / 883H / 894R, 106P / 150S / 218S / 331A / 414G / 486E / 527D / 733E / 749E / 793K, 106P / 150S / 218S / 414G / 486E / 642F / 750P / 793K / 883H,106P / 150S / 218S / 414G / 486E / 750P / 793K / 894R、106P / 150S / 218S / 414G / 527D / 749E / 750P / 883H、106P / 150S / 218S / 414G / 527D / 749E / 793K / 883H / 894G、106P / 150S / 218S / 414G / 749E / 750P / 793K / 883H / 894R、106P / 150S / 218S / 414G / 749E / 793K / 883H、106P / 150S / 218S / 486E / 527D / 749E / 894R、106P / 150S / 218S / 486E / 793K / 883H、106P / 150S / 218S / 527D / 749E / 750P / 793K、106P / 150S / 218S / 527D / 793K / 894G、106P / 150S / 218S / 749E / 750P / 793K、106P / 150S / 218S / 793K、106P / 150S / 218S / 793K / 894R、106P / 150S / 245S / 793K / 883H / 894R、106P / 150S / 414G / 749E / 750P / 793K / 894R、106P / 150S / 414G / 749E / 793K / 894R、106P / 150S / 486E / 527D / 750P / 793K、106P / 150S / 486E / 749E / 793K / 883H / 894G、106P / 150S / 749E / 793K / 883H、106P / 169S / 185G / 218S / 414G / 749E / 750P / 793K、106P / 191R / 280D / 402A / 414G / 444P / 727P、106P / 191R / 414G / 444P / 522V / 928T / 944S、106P / 191R / 414G / 489D / 928T / 944S、106P / 280D / 402A / 414G / 444P / 489D / 727P / 944S、150S / 169S / 218S / 414G / 527D / 793K、150S / 218S / 414G / 486A / 750P / 793K、150S / 218S / 414G / 486E / 749E / 750P、150S / 218S / 414G / 486E / 750P / 793K / 883H、150S / 218S / 414G / 749E / 750P / 793K / 894R、150S / 218S / 414G / 749E / 793K、150S / 218S / 527D / 749E / 793K、150S / 218S / 749E / 750P / 793K、150S / 218S / 749E / 793K、150S / 414G / 486E / 527D / 750P / 894R、150S / 414G / 486E / 749E / 750P / 793K、150S / 486E / 750P / 883H / 894G、169S / 486E / 750P / 793K / 883H、180H / 275M / 402A / 518V / 547G / 610R / 638I / 669H / 671N、180H / 402A / 431V / 507L / 547G / 610R / 669H / 671N / 793G、180H / 402A / 507L / 547G / 610R / 671N、191R / 280D / 402A / 414G / 444P / 465E / 842S / 928T、191R / 280D / 402A / 414G / 444P / 489D / 500A / 944S、191R / 280D / 414G / 444P / 489D / 500A / 522V / 842S / 928T / 944S、191R / 280D / 414G / 444P / 489D / 522V / 727P / 944S、191R / 280D / 414G / 489D / 842S / 928T / 944S、191R / 280D / 414G / 944S、191R / 414G / 522V / 842S / 944S、196V / 402A / 431V / 547G / 610R / 638I、218S / 668D / 700F / 869T、224F / 402A / 507L / 518V / 547G / 638I / 668D、269N / 275M / 431V / 518V / 547G / 638I / 668D / 669H、275M / 281V / 402A / 431V / 507L / 518V / 610R / 668D、275M / 281V / 402A / 507L / 518V / 547G / 638I / 669H / 671N、275M / 281V / 402A / 518V / 547G / 610R / 638I / 671N、275M / 281V / 402A / 518V / 547G / 610R / 668D / 669H / 887D、275M / 281V / 402A / 547G / 610R / 638I / 669H / 671N、275M / 281V / 507L / 547G / 669H / 671N、275M / 281V / 610R / 638I / 668D / 669H、275M / 402A / 431V / 507L / 547G / 671N、275M / 402A / 507L / 547G / 610R / 671N、275M / 402A / 547G / 638I / 669H / 671N、275M / 431V / 518V / 547G / 638I / 668D、275M / 431V / 518V / 610R / 638I / 669H / 671N、275M / 431V / 638I、275M / 507L / 547G / 668D / 669H / 671N、275V / 281V / 402A / 431V / 518V / 547G / 610R / 669H / 671N、275V / 281V / 431V / 518V / 547G / 638I / 669H / 671N、275V / 281V / 671N、275V / 377K / 402A / 507L / 518V / 669H / 671N / 715G、275V / 402A / 431V / 518V / 610R / 638I / 669H / 671N / 922L、275V / 402A / 507L / 547G / 610R / 638I / 668D / 669H、275V / 402A / 507L / 547G / 610R / 638I / 669H / 671N、275V / 402A / 547G / 610R / 638I / 669H / 671N、275V / 402A / 638I / 669H / 671N、275V / 431V / 507L / 518V / 547G / 668D / 669H / 671N、275V / 431V / 507L / 518V / 610R / 669H / 671N、275V / 431V / 507L / 547G / 610R / 638I / 671N、275V / 507L / 518V / 547G / 610R / 638I / 668D / 669H、275V / 507L / 518V / 547G / 638I / 669H / 671N、275V / 507L / 547G / 610R / 638I / 669H / 671N、275V / 518V / 671N、280D / 402A / 536I / 928T、281V / 402A / 507L / 518V / 547G / 610R / 638I / 669H / 671N、281V / 402A / 507L / 547G / 638I / 669H / 671N、281V / 402A / 518V / 547G / 610R / 638I / 668D / 669H、281V / 402A / 518V / 547G / 668D、281V / 431V / 507L / 518V / 547G / 610R / 638I / 668D、402A / 431V / 518V / 547G / 610R / 668D、402A / 431V / 518V / 547G / 671N、402A / 431V / 518V / 610R、The following positions include at least one substitution or set of substitutions selected from 402A / 431V / 547G / 638I / 671N, 431V / 507L / 518V / 541E / 547G / 638I / 669H / 671N, 431V / 507L / 518V / 669H / 671N, 507L / 547G / 610R, 507L / 547G / 638I / 669H / 671N, 547G / 610R / 638I / 671N, and 547G / 638I / 668D, where these positions are numbered with reference to Sequence ID No. 2. In some embodiments, the acid alpha-glucosidase is L29Q / L218S / L240I / S668D / H700F / I744V / I869L, L29Q / L240I / A596P / S668D / I869L, L29Q / L240I / A596S / S668D / H700F / I744V / I869T, L29V / L218S / L240I / H700F / I869T, G36R / K106P / T1 50S / L218S / N527D / A750P / R883H / Q894R, K106P / A112S / T150S / L218S / R414G / N527D / E793K / R883H, K106P / T 150S / N169S / L218S / R414G / T486E / N527D / A750P / Q894R, K106P / T150S / N169S / L218S / R414G / T486E / N527D / Q 894R, K106P / T150S / N169S / L218S / R414G / T486E / Q749E / E793K / R883H / Q894R, K106P / T150S / N169S / L218S / R414G / T486E / A750P / E793K / R883H / Q894R, K106P / T150S / N169S / L218S / R414G / T486E / E793K / R883H, K106P / T150S / N169S / L218S / R414G / T486E / Q894R, K106P / T150S / N169S / L218S / R414G / Q749E / A750P / E793K / R883H, K106P / T150S / N169S / L218S / R414G / Q749E / E793K, K106P / T150S / N169S / L218S / R414G / Q749E / E793K / R883H,K106P / T150S / N169S / L218S / T486E / N527D / Q749E / E793K / Q894R、K106P / T150S / N169S / L218S / T486E / Q749E / R883H、K106P / T150S / N169S / L218S / T486E / R883H、K106P / T150S / N169S / L218S / Q749E / P800A、K106P / T150S / N169S / R414G / T486E / Q749E / A750P / R883H、K106P / T150S / N169S / N527D / Q749E / E793、 K / R883H、K106P / T150S / N169S / Q749E / E793K / R883H / Q894R、K106P / T150S / L218S / V331A / R414G / T486E / N527D / D733E / Q749E / E793K、K106P / T150S / L218S / R414G / T486E / L642F / A750P / E793K / R883H、K106P / T150S / L218S / R414G / T486E / A750P / E793K / Q894R、K106P / T150S / L218S / R414G / N527D / Q749E / A750P / R883H、K106P / T150S / L218S / R414G / N527D / Q749E / E793K / R883H / Q894G、K106P / T150S / L218S / R414G / Q749E / A750P / E793K / R883H / Q894R、K106P / T150S / L218S / R414G / Q749E / E793K / R883H、K106P / T150S / L218S / T486E / N527D / Q749E / Q894R、K106P / T150S / L218S / T486E / E793K / R883H、K106P / T150S / L218S / N527D / Q749E / A750P / E793K、K106P / T150S / L218S / N527D / E793K / Q894G、K106P / T150S / L218S / Q749E / A750P / E793K、K106P / T150S / L218S / E793K、K106P / T150S / L218S / E793K / Q894R、K106P / T150S / P245S / E793K / R883H / Q894R、K106P / T150S / R414G / Q749E / A750P / E793K / Q894R、K106P / T150S / R414G / Q749E / E793K / Q894R、K106P / T150S / T486E / N527D / A750P / E793K、K106P / T150S / T486E / Q749E / E793K / R883H / Q894G、K106P / T150S / Q749E / E793K / R883H、K106P / N169S / V185G / L218S / R414G / Q749E / A750P / E793K、K106P / H191R / G280D / S402A / R414G / A444P / S727P、K106P / H191R / R414G / A444P / E522V / D928T / C944S、K106P / H191R / R414G / A489D / D928T / C944S、K106P / G280D / S402A / R414G / A444P / A489D / S727P / C944S、T150S / N169S / L218S / R414G / N527D / E793K、T150S / L218S / R414G / T486A / A750P / E793K、T150S / L218S / R414G / T486E / Q749E / A750P、T150S / L218S / R414G / T486E / A750P / E793K / R883H、T150S / L218S / R414G / Q749E / A750P / E793K / Q894R、T150S / L218S / R414G / Q749E / E793K、T150S / L218S / N527D / Q749E / E793K、T150S / L218S / Q749E / A750P / E793K、T150S / L218S / Q749E / E793K、T150S / R414G / T486E / N527D / A750P / Q894R、T150S / R414G / T486E / Q749E / A750P / E793K、T150S / T486E / A750P / R883H / Q894G、N169S / T486E / A750P / E793K / R883H、N180H / L275M / S402A / I518V / A547G / W610R / V638I / L669H / S671N、N180H / S402A / M431V / M507L / A547G / W610R / L669H / S671N / E793G、N180H / S402A / M507L / A547G / W610R / S671N、H191R / G280D / S402A / R414G / A444P / G465E / G842S / D928T、H191R / G280D / S402A / R414G / A444P / A489D / D500A / C944S、H191R / G280D / R414G / A444P / A489D / D500A / E522V / G842S / D928T / C944S、H191R / G280D / R414G / A444P / A489D / E522V / S727P / C944S、H191R / G280D / R414G / A489D / G842S / D928T / C944S、H191R / G280D / R414G / C944S、H191R / R414G / E522V / G842S / C944S、A196V / S402A / M431V / A547G / W610R / V638I、L218S / S668D / H700F / I869T、L224F / S402A / M507L / I518V / A547G / V638I / S668D、T269N / L275M / M431V / I518V / A547G / V638I / S668D / L669H、L275M / A281V / S402A / M431V / M507L / I518V / W610R / S668D、L275M / A281V / S402A / M507L / I518V / A547G / V638I / L669H / S671N、L275M / A281V / S402A / I518V / A547G / W610R / V638I / S671N、L275M / A281V / S402A / I518V / A547G / W610R / S668D / L669H / E887D、L275M / A281V / S402A / A547G / W610R / V638I / L669H / S671N、L275M / A281V / M507L / A547G / L669H / S671N、L275M / A281V / W610R / V638I / S668D / L669H、L275M / S402A / M431V / M507L / A547G / S671N、L275M / S402A / M507L / A547G / W610R / S671N、L275M / S402A / A547G / V638I / L669H / S671N、L275M / M431V / I518V / A547G / V638I / S668D、L275M / M431V / I518V / W610R / V638I / L669H / S671N、L275M / M431V / V638I、L275M / M507L / A547G / S668D / L669H / S671N、L275V / A281V / S402A / M431V / I518V / A547G / W610R / L669H / S671N、L275V / A281V / M431V / I518V / A547G / V638I / L669H / S671N、L275V / A281V / S671N、L275V / R377K / S402A / M507L / I518V / L669H / S671N / V715G、L275V / S402A / M431V / I518V / W610R / V638I / L669H / S671N / P922L、L275V / S402A / M507L / A547G / W610R / V638I / S668D / L669H、L275V / S402A / M507L / A547G / W610R / V638I / L669H / S671N、L275V / S402A / A547G / W610R / V638I / L669H / S671N、L275V / S402A / V638I / L669H / S671N、L275V / M431V / M507L / I518V / A547G / S668D / L669H / S671N、L275V / M431V / M507L / I518V / W610R / L669H / S671N、L275V / M431V / M507L / A547G / W610R / V638I / S671N、L275V / M507L / I518V / A547G / W610R / V638I / S668D / L669H、L275V / M507L / I518V / A547G / V638I / L669H / S671N、L275V / M507L / A547G / W610R / V638I / L669H / S671N、L275V / I518V / S671N、G280D / S402A / V536I / D928T、A281V / S402A / M507L / I518V / A547G / W610R / V638I / L669H / S671N、A281V / S402A / M507L / A547G / V638I / L669H / S671N、A281V / S402A / I518V / A547G / W610R / V638I / S668D / L669H、A281V / S402A / I518V / A547G / S668D、A281V / M431V / M507L / I518V / A547G / W610R / V638I / S668D、S402A / M431V / I518V / A547G / W610R / S668D、S402A / M431V / I518V / A547G / S671N、S402A / M431V / I518V / W610R、S402A / M431V / A547G / V638I / S671N、M431V / M507L / I518V / G541E / A547G / V638I / L669H / S671N、M431V / M507L / I518V / L669H / S671N、M507L / A547G / W610R、M507L / A547G / V638I / L669H / S671N、A547G / W610R / V638I / S671N、and includes at least one substitution or set of substitutions at one or more positions selected from A547G / V638I / S668D, where these positions are numbered with reference to Sequence ID No. 2.

[0143] The present invention provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 8. The present invention provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 8. In some embodiments, acid alpha-glucosidase is 4, 27, 27 / 28 / 489, 27 / 418 / 478, 28, 28 / 29, 28 / 29 / 113 / 135 / 138, 28 / 29 / 113 / 135 / 418, 28 / 29 / 135, 28 / 29 / 418, 29 / 113 / 126 / 135 / 193, 2 9 / 113 / 135, 29 / 113 / 135 / 455, 29 / 113 / 138, 29 / 148, 29 / 478, 106, 106 / 138 / 218 / 431 / 671 / 749, 106 / 218 / 281, 106 / 218 / 455, 106 / 218 / 455 / 507 / 749, 106 / 489 / 671, 106 / 638, 106 / 671 / 934, 113, 113 / 135 / 418, 113 / 418 / 455 / 478 / 581, 113 / 418 / 478 / 489 / 581, 135, 135 / 148 / 150 / 418, 135 / 478 / 489 / 581, 135 / 489, 135 / 944, 138 / 218 / 668 / 67 1, 138 / 218 / 749 / 934, 138 / 671 / 749 / 934, 157, 218, 218 / 281, 218 / 281 / 431, 218 / 281 / 671, 218 / 431, 218 / 431 / 489 / 507 / 749 / 934, 218 / 455, 218 / 507 / 749, 218 / 507 / 934,The sequence includes at least one substitution in a position or set of positions selected from 218 / 638 / 671, 218 / 749, 281 / 431 / 489 / 668, 345 / 934, 418, 418 / 489, 431 / 668 / 671, 489 / 638 / 934, 489 / 671 / 934, 489 / 749, 489 / 934, 507 / 668, 507 / 671 / 934, 671 / 749, 671 / 934, and 749 / 784, where these positions are numbered with reference to sequence number 8. In some embodiments, acid alpha-glucosidase is 4H, 27P / 28S / 489R, 27P / 418E / 478T, 27R, 28S, 28S / 29T, 28S / 29T / 113S / 135Q / 138A, 28S / 29T / 113S / 135Q / 418E, 28S / 29T / 135Q, 28S / 29T / 418E, 29T / 113S / 126Q / 135Q / 193Q, 29T / 113S / 135Q, 29T / 113S / 13 5Q / 455V, 29T / 113S / 138A, 29T / 148G, 29T / 478T, 106P, 106P / 138A / 218S / 431V / 671N / 749E, 106P / 218S / 281V, 106P / 2 18S / 455V, 106P / 218S / 455V / 507L / 749E, 106P / 489R / 671N, 106P / 638I, 106P / 671N / 934R, 113S, 113S / 135Q / 418E, 113 S / 418E / 455V / 478T / 581T, 113S / 418E / 478T / 489R / 581T, 135P / 944Y, 135Q, 135Q / 148G / 150G / 418E, 135Q / 478T / 489R / 581T, 135Q / 489R, 138A / 218S / 668D / 671N, 138A / 218S / 749E / 934R, 138A / 671N / 749E / 934R, 157M, 218S, 218S / 281V, 2 18S / 281V / 431V, 218S / 281V / 671N, 218S / 431V, 218S / 431V / 489R / 507L / 749E / 934R, 218S / 455V, 218S / 507L / 749E, 218 S / 507L / 934R, 218S / 638I / 671N, 218S / 749E, 281V / 431V / 489R / 668D, 345K / 934R, 418E, 418E / 489R, 431V / 668D / 671N,It includes at least one substitution or set of substitutions at one or more positions selected from 489R / 638I / 934R, 489R / 671N / 934R, 489R / 749E, 489R / 934R, 507L / 668D, 507L / 671N / 934R, 671N / 749E, 671N / 934R, and 749E / 784T, where these positions are numbered with reference to Sequence ID No. 8. In some embodiments, acidic alpha-glucosidase is P4H, F27P / L28S / A489R, F27P / A418E / A478T, F27R, L28S, L28S / L29T, L28S / L29T / Q113S / S135Q / M138A, L28S / L29T / Q113S / S135Q / A418E, L28S / L29T / S135Q, L28S / L29T / A418E, L29T / Q113S / P126Q / S135Q / H193Q, L 29T / Q113S / S135Q, L29T / Q113S / S135Q / R455V, L29T / Q113S / M138A, L29T / T148G, L29T / A478T, K106P, K106P / M138A / L218S / M431V / S671N / Q749E, K106P / L218S / A281V, K106P / L218S / R455V, K106P / L218S / R455V / M507L / Q749E, K106P / A489R / S671 N, K106P / V638I, K106P / S671N / L934R, Q113S, Q113S / S135Q / A418E, Q113S / A418E / R455V / A478T / K581T, Q113S / A418E / A47 8T / A489R / K581T, S135P / C944Y, S135Q, S135Q / T148G / S150G / A418E, S135Q / A478T / A489R / K581T, S135Q / A489R, M138A / L2 18S / S668D / S671N, M138A / L218S / Q749E / L934R, M138A / S671N / Q749E / L934R, L157M, L218S, L218S / A281V, L218S / A281V / M 431V, L218S / A281V / S671N, L218S / M431V, L218S / M431V / A489R / M507L / Q749E / L934R, L218S / R455V, L218S / M507L / Q749E,L218S / M507L / L934R, L218S / V638I / S671N, L218S / Q749E, A281V / M431V / A489R / S668D, Q345K / L 934R, A418E, A418E / A489R, M431V / S668D / S671N, A489R / V638I / L934R, A489R / S671N / L934R, A4 It includes at least one substitution or set of substitutions at one or more positions selected from 89R / Q749E, A489R / L934R, M507L / S668D, M507L / S671N / L934R, S671N / Q749E, S671N / L934R, and Q749E / A784, where these positions are numbered with reference to Sequence ID 8.

[0144] The present invention provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 14. The present invention provides recombinant acid alpha-glucosidase and / or bioactive recombinant acid alpha-glucosidase fragments comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 14. In some embodiments, acid alpha-glucosidase is 22, 24, 27, 27 / 165, 30, 33, 34, 37 / 62, 37 / 62 / 79 / 196 / 696 / 862, 37 / 62 / 523, 37 / 62 / 523 / 793, 37 / 64 / 66 / 79 / 154 / 523 / 681 / 793 / 862, 37 / 7 9 / 154 / 793, 37 / 196, 37 / 528 / 696 / 793, 37 / 528 / 790, 37 / 528 / 790 / 793 / 862, 37 / 790 / 793, 39, 39 / 58 / 489 / 725 / 830 / 842 / 930 / 944, 39 / 70 / 109 / 830 / 842, 39 / 70 / 489 / 612, 3 9 / 70 / 725, 39 / 267, 39 / 267 / 489 / 522 / 612 / 830 / 842, 39 / 267 / 489 / 830 / 944, 39 / 489 / 500 / 612, 39 / 500 / 612, 40, 44 / 157, 47, 49, 50, 55, 60 / 500 / 612, 62 / 79 / 154 / 862, 62 / 7 9 / 196 / 681 / 862, 62 / 79 / 523 / 528 / 790, 62 / 79 / 790 / 793, 62 / 79 / 862, 62 / 92, 62 / 92 / 790 / 793, 62 / 106 / 523 / 528 / 696 / 793 / 862, 62 / 154 / 696 / 793 / 862, 62 / 793 / 862, 68, 70,70 / 267 / 725 / 944, 70 / 267 / 930 / 944, 70 / 489 / 930, 70 / 725 / 830 / 860 / 930 / 944, 77, 79 / 154 / 681, 79 / 154 / 793 / 862, 79 / 862, 89, 97, 106 / 154, 107, 109, 109 / 522 / 612 / 725, 109 / 522 / 830 / 944, 109 / 612, 118, 149, 157, 158, 178, 179, 196 / 528 / 681 / 790 / 793, 207, 208, 217, 267 / 489 / 500 / 725 / 830 / 930, 267 / 522 / 725, 352, 385, 424, 4 The sequence includes at least one substitution in a position or set of positions selected from 48, 463, 489 / 830 / 944, 500, 500 / 612 / 830 / 860, 500 / 860 / 930, 500 / 930 / 944, 522 / 725, 523, 523 / 790 / 793, 528 / 681, 528 / 793, 528 / 862, 672, 673, 725, 734, 740, 753, 774, 778, 793, 830, 844, 862, 875, 880, 892, 902, 922, 925, 930, 932, 934, 938, and 944, where these positions are numbered with reference to Sequence ID No. 14. In some embodiments, acid alpha-glucosidase is 22R, 24E, 24R, 24W, 27A, 27G, 27G / 165I, 27K, 27R, 27S, 27V, 27W, 30D, 30L, 33G, 33P, 34D, 34M, 34T, 37F / 62E, 37F / 62E / 79S / 196T / 696S / 862Q, 37F / 62E / 523N, 37F / 62E / 523N / 793K, 37F / 64Q / 66G / 79S / 154R / 523N / 681Q / 793K / 862Q, 37F / 79S / 154R / 79 3K, 37F / 196T, 37F / 528S / 696S / 793K, 37F / 528S / 790V, 37F / 528S / 790V / 793K / 862Q, 37F / 790V / 793K, 39D, 39H, 39Q, 39Q / 58L / 489D / 725E / 83 0K / 842S / 930P / 944S, 39Q / 70A / 109P / 830K / 842S, 39Q / 70A / 489D / 612D, 39Q / 70A / 725E, 39Q / 267K, 39Q / 267K / 489D / 522V / 612D / 830K / 842S,39Q / 267K / 489D / 830K / 944S、39Q / 489D / 500A / 612D、39Q / 500A / 612D、40W、44I / 157V、47G、47R、49A、49G、50G、50L、50V、55C、55L、60V / 500A / 612D、62E / 79S / 154R / 862Q、62E / 79S / 196T / 681Q / 862Q、62E / 79S / 523N / 528S / 790V、62E / 79S / 790V / 793K、62E / 79S / 862Q、62E / 92R、62E / 92R / 790V / 793K、62E / 106R / 523N / 528S / 696S / 793K / 862Q、62E / 154R / 696S / 793K / 862Q、62E / 793K / 862Q、68N、68S、68W、70A / 267K / 725E / 944S、70A / 267K / 930P / 944S、70A / 489D / 930P、70A / 725E / 830K / 860F / 930P / 944S、70Q、77W、79S / 154R / 681Q、79S / 154R / 793K / 862Q、79S / 862Q、89R、97D、97G、106R / 154R、107G、109D、109P / 522V / 612D / 725E、109P / 522V / 830K / 944S、109P / 612D、118F、149R、157Q、158E、158F、178G、178V、179L、196T / 528S / 681Q / 790V / 793K、207R、207Y、208G、208I、217A、217D、267K / 489D / 500A / 725E / 830K / 930P、267K / 522V / 725E、352K、352V、385G、424K、448L、463A、489D / 830K / 944S、500A、500A / 612D / 830K / 860F、500A / 860F / 930P、500A / 930P / 944S、522V / 725E、523N、523N / 790V / 793K、528S / 681Q、528S / 793K、528S / 862Q、672E、672K、673N、673R、725F、725V、734K、740G、740Q、753S、774G、774S、778Q、793K、830V、844R、862Q、875D、880R、892L、902L、922E、925A、925W、930P、932A、934F、938A、938P、944R、and include at least one substitution or set of substitutions at one or more positions selected from 944S, where these positions are numbered with reference to sequence number 14. In some embodiments, the acid alpha-glucosidase is I22R, L24E, L24R, L24W, F27A, F27G, F27G / M165I, F27K, F27R, F27S, F27V, F27W, V30D, V30L, E33G, E33P, L34D, L34M, L34T, S37F / A62E, S37F / A62E / N79S / A196T / A696S / R862Q, S37F / A62E / D523N, S37F / A62E / D523N / E793K, S37F / P64Q / R66G / N79S / K1 54R / D523N / E681Q / E793K / R862Q, S37F / N79S / K154R / E793K, S37F / A196T, S37F / N528S / A696S / E793K, S37F / N528S / I790V, S37F / N528S / I79 0V / E793K / R862Q, S37F / I790V / E793K, P39D, P39H, P39Q, P39Q / R58L / A489D / K725E / Q830K / G842S / C930P / C944S, P39Q / V70A / L109P / Q830K / G842S, P39Q / V70A / A489D / S612D, P39Q / V70A / K725E, P39Q / R267K, P39Q / R267K / A489D / E522V / S612D / Q830K / G842S, P39Q / R267K / A489D / Q8 30K / C944S, P39Q / A489D / D500A / S612D, P39Q / D500A / S612D, V40W, T44I / L157V, A47G, A47R, Q49A, Q49G, Q50G, Q50L, Q50V, P55C, P55L, A60V / D500A / S612D, A62E / N79S / K154R / R862Q, A62E / N79S / A196T / E681Q / R862Q, A62E / N79S / D523N / N528S / I790V, A62E / N79S / I790V / E793K, A6 2E / N79S / R862Q, A62E / Q92R, A62E / Q92R / I790V / E793K, A62E / K106R / D 523N / N528S / A696S / E793K / R862Q, A62E / K154R / A696S / E793K / R862Q,A62E / E793K / R862Q, R68N, R68S, R68W, V70A / R267K / K725E / C944S, V70A / R267K / C930P / C944S, V70A / A489D / C930P, V70A / K725E / Q830K / L 860F / C930P / C944S, V70Q, P77W, N79S / K154R / E681Q, N79S / K154R / E793K / R862Q, N79S / R862Q, A89R, A97D, A97G, K106R / K154R, Q107G, L10 9D, L109P / E522V / S612D / K725E, L109P / E522V / Q830K / C944S, L109P / S612D, W118F, P149R, L157Q, T158E, T158F, P178G, P178V, A179L, A1 96T / N528S / E681Q / I790V / E793K, E207R, E207Y, E208G, E208I, Q217A, Q217D, R267K / A489D / D500A / K725E / Q830K / C930P, R267K / E522V / K7 25E, Y352K, Y352V, R385G, H424K, R448L, E463A, A489D / Q830K / C944S, D500A, D500A / S612D / Q830K / L860F, D500A / L860F / C930P, D500A / C 930P / C944S, E522V / K725E, D523N, D523N / I790V / E793K, N528S / E681Q, N528S / E793K, N528S / R862Q, L672E, L672K, P673N, P673R, K725F, K It includes at least one substitution or set of substitutions at one or more positions selected from 725V, H734K, E740G, E740Q, A753S, A774G, A774S, L778Q, E793K, Q830V, E844R, R862Q, N875D, E880R, Q892L, A902L, P922E, K925A, K925W, C930P, S932A, L934F, Q938A, Q938P, C944R, and C944S, where these positions are numbered with reference to Sequence ID No. 14.

[0145] In some embodiments, recombinant acid alpha-glucosidase contains at least one mutation at at least one position, as provided in Tables 3-1, 3-2, 4-1, 6-1, 10-1, 10-2, 12-1, 13-1, 13-2, 14-1, 14-2, 15-1, 16-1, 17-1, 17-2, 17-3, 17-4, 17-5, 17-6, 17-7, 17-8, and / or 17-9. In some additional embodiments, recombinant acid alpha-glucosidase is derived from human acid alpha-glucosidase. In some further additional embodiments, recombinant acid alpha-glucosidase contains the polypeptide sequence of SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104.

[0146] In some embodiments, the polynucleotide encoding the manipulated GAA polypeptide comprises a polynucleotide sequence selected from the polynucleotide sequences encoding SEQ ID NOs: 1, 5, 7, 11, 13, 15, 17, 19, 945, 1955, 2495, 2879 and / or 3103. In some embodiments, the polynucleotide encoding the manipulated GAA polypeptide has at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% nucleotide residue identity with respect to SEQ ID NOs: 1, 5, 7, 11, 13, 15, 17, 19, 945, 1955, 2495, 2879 and / or 3103. In some embodiments, the polynucleotide encoding the manipulated GAA polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 945, 1955, 2495, 2879 and / or 3103 nucleotide residue identity. In some embodiments, the polynucleotide encoding the manipulated GAA polypeptide includes SEQ ID NOs: 1, 5, 7, 11, 13, 15, 17, 19, 945, 1955, 2495, 2879 and / or 3103. In some embodiments, the polynucleotide encoding the manipulated GAA polypeptide consists of SEQ ID NOs: 1, 5, 7, 11, 13, 15, 17, 19, 945, 1955, 2495, 2879 and / or 3103. In some embodiments, a polynucleotide can be hybridized under highly stringent conditions with a reference polynucleotide sequence selected from SEQ ID NOs: 1, 5, 7, 11, 13, 15, 17, 19, 945, 1955, 2495, 2879 and / or 3103, or a complementary sequence thereof, or a polynucleotide sequence encoding any of the variant GAA polypeptides provided herein.

[0147] In some embodiments, an isolated polynucleotide encoding one of the manipulated GAA polypeptides provided herein is manipulated in various ways to result in polypeptide expression. In some embodiments, the polynucleotide encoding the polypeptide is provided as an expression vector in which one or more regulatory sequences are present to modulate the expression of the polynucleotide and / or polypeptide. Manipulation of the isolated polynucleotide prior to its insertion into the vector may be desirable or necessary, depending on the expression vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art.

[0148] In some embodiments, the regulatory sequence includes, among many other sequences, a promoter, a Kozak sequence, a leader sequence, a polyadenylated sequence, a propeptide sequence, a signal peptide sequence, a DNA-based regulatory element for gene therapy retention, and a transcription terminator. As is well known in the art, a suitable promoter may be selected based on the host cell being used. Suitable promoters for directing the transcription of the nucleic acid constructs of this invention in bacterial host cells include the E. coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levanscuase gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltose-producing amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, as well as prokaryotic beta-lactamase genes (see, for example, Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75: 3727-3731

[1978] ), and the tac promoter (see, for example, DeBoer et al.) See, al., Proc. Natl Acad. Sci. USA 80: 21-25

[1983] . (Examples include, but are not limited to, these.)Exemplary promoters for filamentous fungal host cells include promoters derived from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartate proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, for example, WO96 / 00787), as well as the NA2-tpi promoter (promoter from the gene for Aspergillus niger neutral alpha-amylase and Aspergillus Examples include promoter hybrids from the gene for oryzae triose phosphate isomerase, as well as mutant, cleaved, and hybrid promoters of these. Exemplary yeast cell promoters may be derived from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (see, for example, Romanos et al., Yeast 8:423-488

[1992] ).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 vacuolated virus 40 (SV40), Homo sapiens phosphoglycerate kinase, β-actin, elongation factor-1a or glyceraldehyde-3-phosphate dehydrogenase, or Gallus gallus β-actin.

[0149] In some embodiments, the control sequence is a preferred transcriptional terminator sequence, which is a sequence recognized by the host cell for terminating transcription. The terminator sequence is operably ligated to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that functions in a selected host cell is used in the present invention. For example, exemplary transcriptional terminators for filamentous fungal host cells can be obtained from the genes of Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, for example, Romanos et al., cited above). Exemplary terminators for mammalian cells include, but are not limited to, those from cytomegalovirus (CMV), monkey vacuolated virus 40 (SV40), Homo sapiens growth hormone hGH, bovine growth hormone BGH, and human or rabbit betaglobulin.

[0150] In some embodiments, the regulatory sequence is a suitable 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'-exonuclease degradation, and confer resistance to decapping. 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 can be used. Exemplary leaders for filamentous fungal host cells are obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells include, but are not limited to, those derived from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceroaldehyde-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.

[0151] In some embodiments, the control sequence includes a 3' untranslated nucleic acid region and a polyadenylated tail nucleic acid sequence, which are sequences operably ligated to the 3' end of a protein-coding nucleic acid sequence and mediate binding to proteins involved in mRNA transport and translation, as well as mRNA half-life. Any polyadenylated sequence and 3'UTR that function in selected host cells can be used in the present invention. Exemplary polyadenylated sequences for filamentous fungal host cells include, but are not limited to, those from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are also known in the art (see, for example, Guo and Sherman, Mol. Cell. Biol., 15:5983-5990

[1995] ). 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 contain several sequence elements that improve mRNA stability and increase translation.

[0152] 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, directing the coded polypeptide to the cell's secretory pathway. The 5' end of the coding sequence of the nucleic acid sequence may originally contain a signal peptide coding region that is naturally linked within the translational reading frame to a segment of the coding region encoding the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region heterogeneous to the coding sequence. Any signal peptide coding region that directs the expressed polypeptide to the secretory pathway of a selected host cell is used for the expression of the engineered GAA polypeptide provided herein. Effective signal peptide coding regions for bacterial host cells include, but are not limited to, those derived from the genes of Bacillus NClB 11837 maltose-producing amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are known in the art (see, for example, Simonen and Palva, Microbiol. Rev., 57:109-137

[1993] ). Effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, those derived from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartate proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.Useful signal peptides for yeast host cells include, but are not limited to, those derived from the Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase genes. In some embodiments, S. cerevisiae α-conjugation factor prepropeptide (Mfalpha) is used (e.g., SEQ ID NOs. 3383 and 3384). Useful signal peptides for mammalian host cells include, but are not limited to, those derived from the immunoglobulin gamma (IgG) gene. Additional signal peptides useful for mammalian hosts include mouse signal peptides. In some embodiments, synthetic mouse IG signal peptides are used (e.g., SEQ ID NOs. 3381 and 3382).

[0153] In some embodiments, the control sequence is a propeptide coding region that encodes an amino acid sequence located at the amino terminus of the polypeptide. The resulting polypeptide is, in some cases, called a “proenzyme,” “propeptide,” or “zymogen.” By catalytic or autocatalytic cleavage of the propeptide from the propeptide, the propeptide can be converted into a mature, active polypeptide. Propeptide coding regions include, but are not limited to, the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha factor, Rhizomucor miehei aspartate proteinase, and Myceliophthora thermophila lactase (see, e.g., WO95 / 33836). When both the signal peptide region and the propeptide region are located at the amino terminus of the polypeptide, the propeptide region is located adjacent to the amino terminus of the polypeptide, and the signal peptide region is located adjacent to the amino terminus of the propeptide region.

[0154] In some embodiments, regulatory sequences are also utilized. These sequences facilitate the regulation of polypeptide expression in relation to host cell proliferation. 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 Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter.

[0155] In another embodiment, the present invention also provides a recombinant expression vector comprising a polynucleotide encoding an engineered GAA polypeptide and one or more expression regulatory regions, such as promoters and terminators, origins of replication, etc., depending on the type of host to be introduced. In some embodiments, the various nucleic acids and regulatory sequences described above are linked together to produce a recombinant expression vector comprising one or more convenient restriction sites to allow insertion or substitution of the nucleic acid sequence encoding the variant GAA polypeptide at such sites. Alternatively, the polynucleotide sequence of the present invention is expressed by inserting the polynucleotide sequence, or a nucleic acid construct comprising the polynucleotide sequence, into a suitable vector for expression. When creating an expression vector, the coding sequence is positioned within the vector such that the coding sequence is operably linked to a suitable regulatory sequence for expression.

[0156] The recombinant expression vector can be any suitable vector (e.g., adenovirus (AV), adeno-associated virus (AAV), lentivirus (LV), and non-viral vectors, including but not limited to liposomes, plasmids, or viruses) that can be conveniently subjected to recombinant DNA procedures and thereby produce expression of the variant GAA polynucleotide sequence. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector will be introduced. The vector may be a linear plasmid or a closed circular plasmid. In practice, the present invention is not intended to be limited to any particular vector.

[0157] In some embodiments, the expression vector is a self-replicating vector (i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome). The vector may contain some means to ensure self-replication. In some alternative embodiments, the vector may, upon introduction into a host cell, be integrated into the genome and replicate itself along with the chromosome into which it is integrated. Furthermore, a single vector or plasmid may be used, or two or more vectors or plasmids containing total DNA that, when combined, will be introduced into the host cell genome or transposons.

[0158] In some embodiments, the expression vector is pDH. A plasmid map of this vector containing SEQ ID NO: 1 is provided in Figure 9. The sequence of this plasmid is provided as SEQ ID NO: 3379. A plasmid map of this vector containing a beta-lactamase stuffer sequence is provided in Figure 10. The sequence of this plasmid is provided as SEQ ID NO: 3380. The pDH vector is intended to be used for the expression of a variety of genes, including but not limited to polynucleotide sequences encoding acid alpha-glucosidases provided herein. In practice, the stuffer sequence (or SEQ ID NO: 1, present in SEQ ID NO: 3379) is intended to be replaced with any suitable gene of interest.

[0159] In some embodiments, the expression vector preferably contains one or more selectable markers that allow for easy selection of transformed cells. A “selectable marker” is a gene whose product confers biocide or viral resistance, heavy metal resistance, prototrophicity to trophic requirement strains, and similar effects. Examples of bacterial selectable markers include, but are not limited to, 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 include, but are not limited to, 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), argB (ornithine carbamoyltransferase), bar (phosphinotrysin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (adenylate sulfate transferase), and trpC (anthranilate synthase), as well as their equivalents. In another embodiment, the present invention provides a host cell comprising a polynucleotide encoding at least one engineered GAA polypeptide of the present application, wherein the polynucleotide is operably linked to one or more regulatory sequences for the expression of the engineered GAA enzyme in the host cell.Host cells for use in expressing polypeptides encoded by the expression vectors of the present invention are well known in the art and include, but are not limited to, bacterial cells, e.g., E. coli, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells; fungal cells, e.g., yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris [ATCC accession number 201178]); insect cells, e.g., Drosophila S2 and Spodoptera Sf9 cells; animal cells (e.g., CHO, CHO-K1, COS, and BHK); and human cells (e.g., HEK293T, human fibroblasts, THP-1, Jarcutt and Bowes melanoma cell lines); and plant cells.

[0160] Accordingly, in another embodiment, the present invention provides a method for producing an engineered GAA polypeptide, comprising the step of culturing a host cell capable of expressing a polynucleotide encoding the engineered GAA polypeptide under conditions suitable for polypeptide expression. In some embodiments, the method further comprises the step of isolating and / or purifying the GAA polypeptide as described herein.

[0161] Appropriate culture media and growth conditions for the above-mentioned host cells are well known in the art. Polynucleotides for GAA polypeptide expression can be introduced into cells by various methods known in the art. Among the many techniques, electroporation, bioristic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion are particularly noteworthy.

[0162] Engineered GAAs having the properties disclosed herein can be obtained by subjecting polynucleotides encoding naturally occurring or engineered GAA polypeptides to mutagenesis and / or directed evolution methods known in the art and described herein. An exemplary directed evolution technique is mutagenesis and / or DNA shuffling (see, e.g., Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751

[1994] ; 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, among many others, the staggered elongation process (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 16:258-261

[1998] ), mutagenic PCR (see, e.g., Caldwell et al., PCR Methods Appl., 3:S136-S140

[1994] ), and cassette mutagenicity (see, e.g., Black et al., Proc. Natl. Acad. Sci. USA 93:3525-3529

[1996] ).

[0163] For example, mutagenesis and directed evolution methods can be easily applied to polynucleotides to generate variant libraries, which can then be expressed, screened, and assayed. Mutagenesis and directed evolution methods are well known in the art (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, 6,265,201, 6,277,638, 6, No. 287,861, No. 6,287,862, No. 6,291,242, No. 6,297,053, No. 6,303,344, No. 6,309,883, No. 6,319,713, No. 6,319,714, No. 6,323,030, No. 6,326,204, No. 6,335,160, No. 6,335,198, No. 6,344,356, No. 6,352,859, No. 6,355,484, No. 6,358,740, No. 6,358,742, No. 6,365,37 No. 7, No. 6,365,408, No. 6,368,861, No. 6,372,497, No. 6,337,186, No. 6,376,246, No. 6,379,964, No. 6,387,702, No. 6,391,552, No. 6,39 1,640, 6,395,547, 6,406,855, 6,406,910, 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, No. 6,436,675, No. 6,444,468, No. 6,455,253, No. 6,479,652, No. 6,482,647, No. 6,483,011, No. 6,484,105, No. 6,489,146, No. 6,500,617 No. 6,500,639, No. 6,506,602, No. 6,506,603, No. 6,518,065, No. 6,519,065, No. 6,521,453, No. 6,528,311, No. 6,537,746, No. 6,573,No. 098, same as No. 6,576,467, same as No. 6,579,678, same as No. 6,586,182, same as No. 6,602,986, same as No. 6,605,430, same as No. 6,613,514, same as No. 6,653,072, same as No. 6,686,515, same as No. 6,703,240, same as No. 6,716,631, same as No. 6,825,001, same as No. 6,902,922, same as No. 6,917,882, same as No. 6,946,296, same as No. 6,961,664, same as No. 6, No. 995,017, No. 7,024,312, No. 7,058,515, No. 7,105,297, No. 7,148,054, No. 7,220,566, No. 7,288,375, No. 7,384,387, No. 7,421,347, No. 7,430,477, No. 7,462,469, No. 7,534,564, No. 7,620,500, No. 7,620,502, No. 7,629,170, No. 7,702,464, and so on. 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. 7,783,428, No. 7,873,477, No. 7,873,499, No. 7,904,249, No. 7,957,912, No. 7,981,614, No. 8,014,961, No. 8,029,988 Numbers: 8,048,674, 8,058,001, 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, 9,684,771, 9,665.Patent No. 694, as well as WO95 / 22625, WO97 / 0078, WO97 / 35966, WO98 / 27230, WO00 / 42651, WO01 / 75767, WO2009 / 152336, WO2013 / 138339, WO2015 / 048572, and WO2015 / 048573, and all related U.S. and non-U.S. corresponding patents of these listed patents and applications; Ling et al., Anal. Biochem., 254:157-78

[1997] ; Dale et al., Meth. Mol. Biol., 57:369-74

[1996] ; Smith, Ann. Rev. Genet., 19:423-462

[1985] ; Botstein et al. al., Science, 229:1193-1201

[1985] ; Carter, Biochem. J., 237:1-7

[1986] ; Kramer et al., Cell, 38:879-887

[1984] ; Wells et al., Gene, 34:315-323

[1985] ; Minshull et al., Curr. Chem. Biol., 3:284-290

[1999] ; Christians et al., Nat. Biotechnol., 17:259-264

[1999] ; Crameri et al., Nature, 391:288-291

[1998] ; Crameri, et al., Nat. Biotechnol., 15:436-438

[1997] ;Zhang et al., Proc. See also Nat. Acad. Sci. USA, 94:4504-4509

[1997] ; Crameri et al., Nat. Biotechnol., 14:315-319

[1996] ; Stemmer, Nature, 370:389-391

[1994] ; and Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751

[1994] (all of which are incorporated herein by reference).

[0164] In some embodiments, enzyme clones obtained after mutagenesis are screened by subjecting the enzyme to a defined temperature (or other assay conditions) and measuring the amount of residual enzyme activity after heat treatment or other assay conditions. Clones containing polynucleotides encoding the GAA polypeptide are then isolated from the gene, sequenced to identify any nucleotide sequence changes (if any), and used to express the enzyme in host cells. Measurement of enzyme activity from the expression library can be performed using any suitable method known in the art (e.g., standard biochemical techniques such as HPLC analysis).

[0165] For manipulated polypeptides with known sequences, enzyme-encoding polynucleotides can be prepared by standard solid-phase methods according to known synthetic methods. In some embodiments, fragments of up to approximately 100 bases can be synthesized individually and then joined (e.g., by enzymatic or chemical ligation methods, or by polymerase-mediated methods) to form any desired continuous sequence. For example, the polynucleotides and oligonucleotides disclosed herein are usually carried out by automated synthesis methods, but can also be prepared by chemical synthesis using the classical phosphoramidite method (see, e.g., Beaucage et al., Tetra. Lett., 22:1859-69

[1981] ; and Matthes et al., EMBO J., 3:801-05

[1984] ). According to the phosphoramidite method, oligonucleotides are synthesized (e.g., in an automated DNA synthesizer), purified, annealed, ligated, and cloned into a suitable vector.

[0166] Accordingly, in some embodiments, a method for preparing an engineered GAA polypeptide may include the steps of (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the amino acid sequences of any variant provided in Table 3-1, 3-2, 4-1, 6-1, 10-1, 10-2, 12-1, 13-1, 13-2, 14-1, 14-2, 15-1, 16-1, 17-1, 17-2, 17-3, 17-4, 17-5, 17-6, 17-7, 17-8, and / or 17-9, as well as SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104, and (b) expressing the GAA polypeptide encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide may have, as necessary, one or more (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence may have, as necessary, 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 residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence may have, as necessary, 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 residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence may have, as necessary, 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 residue deletions, insertions, and / or substitutions. In some embodiments, substitutions may be conserved substitutions or non-conservative substitutions.

[0167] The expressed and manipulated GAA polypeptide can be measured for any desired improved properties (e.g., activity, selectivity, stability, acid resistance, protease sensitivity, etc.) using any suitable assay known in the art, including but not limited to the assays and conditions described herein.

[0168] In some embodiments, some or all of the manipulated GAA polypeptides expressed in host cells are recovered from the cells and / or culture medium using one or more of the well-known techniques for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography.

[0169] Chromatographic techniques for the isolation of GAA polypeptides include, among others, reverse-phase chromatography, high-performance liquid chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, gel electrophoresis, and affinity chromatography. The conditions for purifying a particular enzyme 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 can be used to isolate improved variant GAA enzymes. In some embodiments utilizing affinity chromatography purification, any antibody that specifically binds to the variant GAA polypeptide is used. For antibody production, various host animals, including but not limited to rabbits, mice, and rats, are immunized by injection of GAA polypeptide (e.g., GAA variant) or fragments thereof. In some embodiments, the GAA polypeptide or fragment is bound to a suitable carrier such as BSA by side-chain functional groups or by linkers bound to side-chain functional groups.

[0170] In some embodiments, the engineered GAA polypeptide is produced in host cells by a method comprising the steps of culturing host cells containing a polynucleotide sequence encoding the engineered GAA polypeptide described herein (e.g., S. cerevisiae, Daucus carota, Nicotiana tabacum, H. sapiens [e.g., HEK293T], or the genus Cricetulus [e.g., CHO]) under conditions that promote the production of the engineered GAA polypeptide, and recovering the engineered GAA polypeptide from the cells and / or culture medium.

[0171] In some embodiments, the engineered GAA polypeptide is produced in host cells by a method comprising the steps of culturing host cells containing a polynucleotide sequence encoding the engineered GAA polypeptide described herein under conditions that promote the production of the engineered GAA polypeptide, and recovering the engineered GAA polypeptide from the cells and / or culture medium.

[0172] In some preferred embodiments, the present invention comprises a method for producing an engineered GAA polypeptide, comprising the steps of culturing recombinant bacterial cells containing a polynucleotide sequence encoding an engineered GAA polypeptide, which, when aligned, has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference sequence SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104, and has one or more amino acid residue differences with SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104, and / or combinations thereof, under culture conditions suitable to enable the production of the engineered GAA polypeptide, and optionally, recovering the engineered GAA polypeptide from the culture and / or cultured bacterial cells.

[0173] In some embodiments, the manipulated GAA polypeptide, once recovered from recombinant host cells or cell cultures, is further purified by any suitable method known in the art. In some additional embodiments, the purified GAA polypeptide is combined with other components and compounds to provide compositions and formulations that appropriately contain the manipulated GAA polypeptide for different applications and uses (e.g., pharmaceutical compositions). Composition:

[0174] The present invention provides an engineered GAA polypeptide suitable for use in pharmaceutical compositions and other compositions, such as health / nutritional supplements, as well as for other purposes. Pharmaceutical composition:

[0175] Depending on the method of administration, the composition containing a therapeutically effective amount of the manipulated GAA according to the present invention may be in the form of a solid, semi-solid, gel, or liquid. In some embodiments, the composition may include other pharmaceutically acceptable components, such as diluents, buffers, excipients, salts, emulsifiers, preservatives, stabilizers, fillers, and other components. Details regarding the formulation and administration techniques are well known in the art and described in the literature.

[0176] In some embodiments, the manipulated GAA polypeptide is formulated for use in oral pharmaceutical compositions. Any form suitable for use when delivering the manipulated GAA polypeptide is used in the present invention, such as pills, tablets, gel tabs, capsules, lozenges, sugar-coated pills, powders, soft gels, sol-gels, gels, emulsions, grafts, patches, sprays, ointments, liniments, creams, pastes, jellies, topical medications, aerosols, chewing gums, lubricants, sticks, suspensions (including, but not limited to, oily suspensions, oil-in-water emulsions, etc.), slurries, syrups, controlled-release formulations, suppositories, etc. In some embodiments, the manipulated GAA polypeptide is provided in a form suitable for injection (i.e., in an injectable formulation). In some embodiments, the manipulated GAA 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 manipulated GAA polypeptide is encapsulated. In some alternative embodiments, the engineered GAA polypeptide is encapsulated in nanostructures (e.g., nanotubes, nanocapsules, nanocapsules, or microcapsules, microspheres, liposomes, etc.). In practice, the invention is not intended to be limited to any particular delivery formulation and / or delivery means. The engineered GAA polypeptide is intended to be administered by any suitable means known in the art, including but not limited to parenteral, oral, topical, transdermal, intranasal, intraocular, intrathecal, and via grafts.

[0177] In some embodiments, the manipulated GAA polypeptide is glycosylated, PEGylated (i.e., modified with polyethylene glycol [PEG] or activated PEG, etc.), or chemically modified with other compounds (see, e.g., Ikeda, Amino Acids 29:283-287

[2005] ; U.S. Patents Nos. 7,531,341, 7,534,595, 7,560,263, and 7,53,653; U.S. Patent Application Publications Nos. 2013 / 0039898 and 2012 / 0177722, etc.). In practice, the present invention is not intended to be limited to any particular delivery method and / or mechanism.

[0178] In some additional embodiments, the engineered GAA polypeptide is provided for delivery to cells or tissues by gene therapy, including but not limited to viral delivery vectors containing adenovirus (AV), adeno-associated virus (AAV), lentivirus (LV), or non-viral vectors (e.g., liposomes). In some embodiments, the engineered GAA polypeptide is provided for delivery to cells or tissues by mRNA therapy after formulation of a polyribonucleotide sequence, via encapsulation delivery such as liposomes. In some additional embodiments, the engineered GAA polypeptide is provided for delivery to cells or tissues by cell therapy, in which the polynucleotide sequence encoding the engineered GAA polypeptide is introduced into exogenous cells, and those cells (or more cells) are introduced into a recipient (e.g., a patient presenting with or at risk of developing Pompe disease).

[0179] In some additional embodiments, the engineered GAA polypeptide is provided in a formulation comprising matrix-stabilized enzyme crystals. In some embodiments, the formulation comprises a crosslinked crystalline engineered GAA enzyme and a polymer having a reactive moiety attached to the enzyme crystal. The present invention also provides engineered GAA polypeptides in polymers.

[0180] In some embodiments, compositions comprising the manipulated GAA polypeptide of the present invention include one or more commonly used carrier compounds, including but not limited to 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., acacia gum, tragacanth gum, guar gum, etc.), and / or proteins (e.g., gelatin, collagen, etc.). Additional components in oral formulations may include colorants and / or sweeteners (e.g., glucose, sucrose, and mannitol), lubricants (e.g., magnesium stearate), and enteric coating agents (e.g., methacrylate polymers, hydroxypropyl methylcellulose phthalates, and / or any other suitable enteric coating agents known in the art). In some embodiments, disintegrants or solubilizers are included (e.g., cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, e.g., sodium alginate). In some embodiments, the manipulated GAA 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 the case of liquid formulations. In some embodiments, the manipulated GAA polypeptide is administered to a subject in combination with other compounds, molecules, and / or materials used in the treatment of Pompe disease, including but not limited to pharmacological chaperones, as well as any other suitable compounds. In some additional embodiments, the pharmaceutical composition is suitable for parenteral injection into humans. In some embodiments, the pharmaceutical composition comprises pills, tablets, capsules, or gel caps further having an enteric coating.

[0181] In some embodiments, the present invention provides engineered GAA polypeptides suitable for use in reducing the concentration of glycogen in tissues. The dosage of engineered GAA polypeptide administered to an animal depends on the disease state, the animal's overall condition, and other factors known to those skilled in the art. In some embodiments, the composition is for single or multiple doses to an animal. In some embodiments, the concentration of engineered GAA polypeptide in the composition administered to an animal (e.g., a human with Pompe disease) is intended to be sufficient to effectively treat, improve, and / or prevent the symptoms of the disease (e.g., Pompe disease and / or conditions, diseases, and / or symptoms associated with Pompe disease). In some embodiments, engineered GAA polypeptides are administered in combination with other pharmaceutical and / or dietary compositions.

[0182] The above-described and other aspects of the present invention may be better understood in relation to the following non-limiting embodiments. These embodiments are provided solely for illustrative purposes and are not intended to limit the scope of the invention in any way. [Examples]

[0183] experiment The following examples, including the experiments and results achieved, are provided solely for illustrative purposes and should not be construed as limiting the invention.

[0184] In the experiments disclosed below, 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); cm (centimeters); mm (millimeters); um and μm (micrometers); sec. (seconds); min (minutes); h and hr (hours); U (units); MW (molecular weight); rpm (revolutions per minute); °C (degrees Celsius); CDS (coded sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coli W3110 (Coli Genetic Stock Center [CGSC], New Commonly used laboratory E. coli strains available from Haven, CT); DPBS (Dulbeccio phosphate buffered saline); LB (Luria-Burtani); TB (Terrific broth); 4-MUGlu (4-methylumbelliferyl α-D-glucopyranoside); SD-Ura (uracil-free single dropout medium); HPLC (high-pressure 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); MeOH (methanol); Axygen (Axygen, Inc., Union City, CA); Athens Research (Athens Research Technology, Athens, GA); ProSpec (ProSpec Tany Technogene, East Brunswick, NJ); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Ram Scientific (Ram Scientific, Inc., Yonkers, NY); Pall Corp. (Pall, Corp., Pt. Washington, NY); Millipore (Millipore, Corp., Billerica MA); Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI); Molecular Devices (Molecular Devices, LLC, Sunnyvale, CA); Kuhner (Adolf Kuhner, AG, Basel, Switzerland); Microfluidics (Microfluidics Corp., Westwood, MA); Thermotron (Thermotron, Inc., Holland, MI); Cambridge Isotope Laboratories (Cambridge Isotope Laboratories, Inc., Tewksbury, MA); Applied Biosystems (Applied Biosystems, a part of Life Technologies, Corp., Grand Island, NY); Greiner Bio-One (Greiner Bio-One North America, Monroe, NC); Agilent (Agilent Technologies, Inc., Santa Clara, CA); Thermo Scientific (a part of Thermo Fisher Scientific, Waltham, MA); Corning (Corning, Inc., Palo Alto, CA); Megazyme (Megazyme International, Wicklow, Ireland); Enzo (Enzo Life Sciences, Inc., Farmingdale, NY); GE Healthcare (GE Healthcare Bio-Sciences, Piscataway, NJ); Pierce (Pierce Biotechnology (now a part of Thermo Fisher Scientific), Rockford, IL); Phenomenex (Phenomenex, Inc., Torrance, CA); Optimal (Optimal Biotech Group, Belmont, CA); and Bio-Rad (Bio-Rad Laboratories, Hercules, CA). (Example 1) GAA gene acquisition and expression vector construction

[0185] This example describes the acquisition and construction of a GAA gene expression vector. A synthetic gene encoding WT human GAA (Uniprot ID P10253) with the native signal peptide removed was designed for optimized gene expression in Saccharomyces cerevisiae and fused to a yeast MFα signal peptide sequence (SEQ ID NO: 3383) to generate the gene sequence represented by SEQ ID NO: 2, which was then cloned into the yeast expression vector pYT-72 as previously described (see, for example, U.S. Patent Application Publication No. 2017 / 0360900A1). Recombinant cloning and gene expression were performed in S. cerevisiae strain INVSc1. A library of gene variants was generated from this plasmid construct using directed evolution techniques (see, for example, U.S. Patent No. 8,383,346 and WO2010 / 144103).

[0186] For secretory expression and transient transfection in mammalian cells, chimeric GAA expression constructs encoding synthetic mouse IG signal peptides (residues 1-19 of Uniprot accession number A0N1R5, SEQ ID NO: 3381) fused to synthetic genes encoding different GAA variants were generated as follows. In some embodiments, the synthetic GAA gene variant is based on a GAA sequence codon-optimized for yeast (SEQ ID NO: 3), while in some alternative embodiments, the synthetic GAA variant is based on a GAA sequence codon-optimized for mammalian expression (SEQ ID NO: 5). Fragments encoding the synthetic mouse IG signal peptides (SEQ ID NOs: 3381 and 3382) and the encoding sequence of the mature form of GAA were amplified using oligonucleotides containing adjacent restriction enzymes to enable cloning to either the BamHI / XhoI or HindIII / XhoI site. For mammalian expression, the PCR product was ligated to either the BamHI / XhoI or HindIII / XhoI site (Invitrogen) of the linearized vector pcDNA3.1(+). Using directed evolution, specific gene variants derived from sequence numbers 18–828 were generated within pcDNA3.1(+) plasmid constructs (see, for example, U.S. Patent No. 8,383,346 and WO2010 / 144103, each of which is incorporated by reference in whole).

[0187] pDH vectors were generated with optimized vector copy number, desired selective drug, and base pair length to enable compatibility with library variant generation. For mammalian expression, PCR products were ligated to either the BamHI / XhoI or HindIII / XhoI site of the linearized pDH vector. Directional evolution was used to generate specific gene variants (sequences 829-3378) derived from SEQ ID NO: 20 within the pDH plasmid construct. A plasmid map for pDH containing WT GAA is shown in Figure 9, and the plasmid sequence is provided as SEQ ID NO: 3379. In addition, a plasmid map for pDH containing a beta-lactamase (bla) stuffer sequence is provided in Figure 10, and the sequence is provided as SEQ ID NO: 3380.

[0188] In some experiments, GAA variant expression was performed using linear PCR amplification products of the above expression cassettes in pcDNA3.1(+) or pDH (i.e., chimeric expression constructs consisting of synthetic mouse IG signal peptides (residues 1-19 of Uniprot accession number A0N1R5; SEQ ID NO: 3381) fused to synthetic genes encoding different GAA variants). PCR amplification was performed using optimizations generally known to those skilled in the art, with primer pairs (SEQ ID NOs: 3387 and 3388) for pcDNA3.1(+) and primer pairs (SEQ ID NOs: 3385 and 3386; or SEQ ID NOs: 3387 and 3388) for pDH. In some cases, phosphorothioate primers were used. (Example 2) High-throughput growth and GAA assay of Saccharomyces cerevisiae

[0189] This example describes experiments including high-throughput proliferation of cells producing GAA variants, and assays for determining GAA activity. High-throughput (HTP) propagation of S. cerevisiae

[0190] Yeast (INVSc1) cells transformed with vectors expressing GAA and GAA variants using a lithium acetate method known in the art were selected on SD-Ura agar plates. After incubation at 30°C for 72 hours, colonies were placed in wells of AXYGEN® 1.1 ml 96-well deep-well plates (Axygen) filled with 200 μl / well of SD-Ura broth (2 g / L SD-Ura, 6.8 g / L yeast nitrogen-based amino acid-free [Sigma Aldrich]) supplemented with 6% glucose, 3.06 g / L sodium dihydrogen phosphate, 0.804 g / L disodium hydrogen phosphate, and pH 6.0. The cells were grown for 20-24 hours in a Kuhner shaker (250 rpm, 30°C, and 85% relative humidity). The overnight culture sample (20 μL) was transferred to a COSTAR® 96-well deep plate (Corning) filled with 380 μL of SD-ura broth supplemented with 2% glucose. The plate was incubated for 66–84 hours in a Kuhner shaker (250 rpm, 30°C, and 85% relative humidity). The cells were then pelleted (4000 rpm × 20 minutes) and the supernatant (conditional medium) was stored at 4°C until analysis. HTP analysis of the supernatant

[0191] The activity of the GAA variant was determined by measuring the hydrolysis of 4-methylumbelliferyl α-D-glucopyranoside (4-MUGlu). For the non-exposure assay, 50 μL of SD-URA medium prepared as described above was mixed with 50 μL of 1 mM 4-MUGlu in McIlvaine buffer (McIlvaine, J. Biol. Chem., 49:183-186

[1921] ), pH 4.5, in a 96-well, black, opaque-bottom plate. The reaction was briefly mixed and incubated at 37°C for 4–24 hours, after which the reaction was stopped with 100 μL of 0.5 M sodium carbonate, pH 10.5. Hydrolysis was analyzed by monitoring fluorescence (Ex. 355 nm, Em. 460 nm) using a SPECTRAMAX® M2 microplate reader (Molecular Devices). HTP analysis of supernatant exposed to neutral buffer solution

[0192] GAA variants were exposed to neutral to near-neutral (pH 6.5–7.5) buffers to simulate the pH the variants would encounter in the blood after administration to patients. First, 50 μL of GAA variant in SD-URA condition medium and 50 μL of McIlbine buffer (pH 6.5–7.4) were added to the wells of a 96-well round-bottom plate. The plate was sealed and incubated at 37°C for 1 hour. Next, 50 μL of each exposed sample was mixed with 50 μL of 1 mM 4-MUGlu in McIlbine buffer, pH 4.4. The reaction was briefly mixed and incubated at 37°C for 4–24 hours, after which the reaction was stopped with 100 μL of 0.5 M sodium carbonate, pH 10.5. Hydrolysis was analyzed by monitoring fluorescence (Ex. 355 nm, Em. 460 nm) using a SPECTRAMAX® M2 microplate reader (Molecular Devices). HTP analysis of supernatant pretreated with acid

[0193] GAA variants were exposed to an acidic (pH 3) buffer to simulate the pH the variant would encounter in lysosomes after administration to a patient. First, 50 μL of GAA variant in SD-URA condition medium and 50 μL of McIlbine buffer (pH 3) were added to the wells of a 96-well round-bottom plate. The plate was sealed and incubated at 37°C for 1 hour. Next, 50 μL of each exposed sample was mixed with 50 μL of 1 mM 4-MUGlu in McIlbine buffer, pH 4.4. The reaction was briefly mixed and incubated at 37°C for 4–24 hours, after which the reaction was stopped with 100 μL of 0.5 M sodium carbonate, pH 10.5. Hydrolysis was analyzed by monitoring fluorescence (Ex. 355 nm, Em. 460 nm) using a SPECTRAMAX® M2 microplate reader (Molecular Devices). HTP analysis of supernatant in glycogen substrate

[0194] The hydrolytic activity of GAA variants was determined by measuring the hydrolysis of glycogen to glucose. For the non-exposure assay, 50 μL of SD-URA medium prepared as described above was mixed with 50 μL of 5 mM glycogen in pH 4.5 McIlvine buffer (7.71 mL of 0.2 M Na2HPO4 and 12.29 mL of 0.1 M citrate) in a 96-well, black, opaque-bottom plate. The reactants were briefly mixed and incubated at 37°C for 24 hours. After incubation, 20 μL of glycogen hydrolysis reaction mixture was mixed with 80 μL of AMPLEX® Red Glucose Assay Kit (Sigma) to determine the glucose content of each reactant. Hydrolysis was analyzed by monitoring fluorescence (Ex. 544 nm, Em. 585 nm) using a SPECTRAMAX® M2 microplate reader (Molecular Devices). (Example 3) GAA and GAA variants

[0195] GAA and GAA variant activity was determined by assaying enzyme activity after a series of independent exposures. Results for all variants and substitutions in each of their polypeptide sequences are reported in relation to Sequence ID No. 2. These variants were tested for GAA 4-MUGlu activity (FIOPC with unexposed activity), after pH 6.5 incubation (FIOPC with pH 6.5 stable activity), and for glycogen hydrolysis (FIOPC with glycogen activity), as described in Example 2. Tables 3-1 and 3-2 provide the results of these assays. [Table 3-1-1] [Table 3-1-2] [Table 3-1-3] [Table 3-2-1] [Table 3-2-2] [Table 3-2-3] [Table 3-2-4] [Table 3-2-5] (Example 4) GAA variant of sequence number 8

[0196] This example describes the analysis of GAA variants derived from Sequence ID No. 8 regarding the improvement of GAA activity after a series of exposures. Directional evolution of GAA encoded by Sequence ID No. 8 was performed by constructing a library of variant genes. These libraries were then seeded and grown as described in Example 2, and screened for GAA 4-MUGlu activity in unexposed, pre-incubation-free activity assays (FIOPC for unexposed activity), after pH 7 incubation (FIOPC for pH 7 stable activity), or after pH 3 incubation (FIOPC for pH 3 stable activity). The results are shown in Table 4-1. [Table 4-1-1] [Table 4-1-2] [Table 4-1-3] (Example 5) GAA assay achieved by high-throughput proliferation of adherent mammalian cells and adherent mammalian expression. High-throughput (HTP) proliferation of GAA and GAA variants in adherent mammalian cells (HEK293T)

[0197] HEK 293T cells were transfected using lipofection with LIPOFECTAMINE® 3000 reagent (ThermoFisher Scientific) to encode synthetic mouse IG signal peptides (SEQ ID NOs. 3381 and 3382) fused to wild-type GAA or a GAA variant, either as pcDNA 3.1(+) vectors (ThermoFisher Scientific), pDH vectors, or PCR-amplified linear DNA (as described in Example 1). HEK 293T cells were cultured in standard complete growth medium (DMEM containing 10% fetal bovine serum [both from Corning]) and placed in NUNC® Edge 2.0 96-well plates (ThermoFisher Scientific) in 0.5 × 10⁶ wells. 5 Cells were seeded at a density of 250 μL / well and allowed to adhere and grow for 24 hours at 37°C in the presence of 5% CO2, after which they were subjected to lipofection-mediated transfection. After transfection, cells were incubated for 24–96 hours to allow expression of the GAA variant and secretion into the conditioning medium. Conditioning medium (20–100 μL) from the HEK293T transfection was then transferred to a new 96-well plate for analysis of activity, stability, or uptake into cells. HTP analysis of the supernatant

[0198] The activity of the GAA variant was determined by measuring the hydrolysis of 4-methylumbelliferyl α-D-glucopyranoside (4-MUGlu). For the non-exposure assay, 20 μL of HEK 293T medium prepared as described above was mixed with 50 μL of 1.5 mM 4-MUGlu in McIlvaine buffer (McIlvaine, J. Biol. Chem., 49:183-186

[1921] ), pH 4.4, in a 96-well, black, opaque-bottom plate. The reaction was incubated at 25–37°C for 30–60 minutes with stirring at 400 rpm, and then stopped with 100 μL of 0.5 M sodium carbonate, pH 10.5. Hydrolysis was analyzed by monitoring fluorescence (Ex. 355 nm, Em. 460 nm) using either a SPECTRAMAX® M2 microplate reader (Molecular Devices) or an ENVISION® microplate reader (Perkin Elmer). HTP analysis of supernatant exposed to neutral buffer solution

[0199] GAA variants were exposed to neutral buffer to simulate the pH the variant would encounter in the blood after administration to a patient. First, 20 μL of conditional medium containing the GAA variant from HEK 293T expression was combined with 100 μL of DPBS buffer (pH 7.4) in a 96-well plate. The plate was sealed and incubated at 37°C for 24–96 hours. Next, 20 μL of the neutral pH-exposed sample was mixed with 50 μL of 1.5 mM 4-MUGlu in McIlbine buffer, pH 4.4. The reaction was incubated at 37°C for 180 minutes with stirring at 400 rpm, and then the reaction was stopped with 100 μL of 0.5 M sodium carbonate, pH 10.5. Hydrolysis was analyzed by monitoring fluorescence (Ex. 355 nm, Em. 460 nm) using a SPECTRAMAX® M2 microplate reader or an ENVISION® microplate reader (Perkin Elmer). HTP analysis of supernatant exposed to plasma

[0200] GAA variants were exposed to plasma to simulate the conditions they would encounter in the blood after administration to a patient. First, 30 μL of conditional medium containing the GAA variant from HEK 293T expression 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 2–4 hours. Next, 10 μL of the plasma exposure sample was mixed with 50 μL of 1.5 mM 4-MUGlu in McIlbine buffer, pH 4.4. The reaction was incubated at 25–37°C for 15–60 minutes with stirring at 400 rpm, and then the reaction was stopped with 100 μL of 0.5 M sodium carbonate, pH 10.5. Hydrolysis was analyzed by monitoring fluorescence (Ex. 355 nm, Em. 460 nm) using a SPECTRAMAX® M2 microplate reader or an ENVISION® microplate reader (Perkin Elmer). HTP analysis of GAA activity in lysates of Pompe fibroblasts and C2C12 GAA knockout myoblasts

[0201] GAA variants produced by HTP were incubated with target cells, and residual intracellular activity was assayed after 24 hours. These experiments used mammalian cells lacking functional GAA activity, namely Pompe patient-derived fibroblasts (Coriell Institute for Medical Research #GM00248) and C2C12 myoblasts in which the native GAA gene was knocked out using Crispr-Cas9 editing. In these experiments, Pompe fibroblasts or C2C12 GAA knockout myoblasts were seeded in black, clear-bottomed, tissue-culture-treated COSTAR® 96-well plates (Corning, 3904) and grown in standard complete growth medium until confluence. Once confluence was reached, the complete growth medium was removed from the plates using an automated BIOMEK® i5 liquid-processing robot. As described above, the conditional medium produced by transiently transfected HEK293T cells was transfected into Pompe patient-derived fibroblasts and C2C12 myoblasts, and incubated for 4–24 hours at 37°C and 5% CO2. The medium was removed from the culture using an automated BIOMEK® i5 liquid processing robot. The cells were briefly washed with 150 μL of 1× DPBS / well, and the DPBS was removed using an automated BIOMEK® i5 liquid processing robot. Next, 200 μL of standard complete growth medium was added to each well, and the plates were returned to the incubator for 0–72 hours. At the completion of incubation, the standard complete growth medium was removed using an automated BIOMEK® i5 liquid processing robot. The cells were washed with 150 μL of 1× DPBS / well, and the DPBS was removed using an automated BIOMEK® i5 liquid processing robot. Cells were lysed by adding 50 μL of McIlbine buffer, pH 4.4, supplemented with 0.2-0.5% TRITON X-100 (trademark) nonionic surfactant (Sigma #93443), and stirring at room temperature for 30 minutes. Activity was assessed by adding 50 μL of 1.5 mM 4-MUGlu to McIlbine buffer, pH 4.4.The plate was sealed and incubated at 37 °C for 300 - 360 minutes while stirring at 400 rpm. After incubation, the reaction was stopped with 100 μL of 0.5 M sodium carbonate, pH 10.5. Hydrolysis was analyzed by monitoring fluorescence (Ex. 355 nm, Em. 460 nm) using a SPECTRAMAX® M2 microplate reader or an ENVISION® microplate reader (Perkin Elmer). The FIOPC of cell uptake was calculated by dividing the intracellular activity of the GAA variant by the activity of the reference polypeptide with the indicated SEQ ID number. (Example 6) GAA variant of SEQ ID NO: 16

[0202] In this example, experiments on the evolution of a GAA variant derived from SEQ ID NO: 16 and screening for improvement of GAA activity after a series of exposures are described. The GAA synthetic gene encoding SEQ ID NO: 13 was fused to a synthetic mouse IG signal peptide polynucleotide (SEQ ID NO: 3381) (as described in Example 1) to generate a synthetic gene encoding SEQ ID NO: 15. Directed evolution of the GAA encoded by SEQ ID NO: 16 was performed by constructing a library of variant genes. These libraries were then seeded, grown, and screened for GAA MU-Glu activity (“FIOPC of non-exposed activity”), and also screened after pH 7.4 pre-incubation (“FIOPC of pH 7.4 stability and activity”), as described in Example 5. Variants were also tested for 4-MUGlu activity after lysis of Pompe fibroblasts (“FIOPC of activity from Pompe fibroblast lysate”) or after lysis of GAA - / - C2C12 cells (“FIOPC of activity from C2C12 GAA - / - lysate”), as described in Example 5. The results of these assays are presented in Table <6-1>.

Table 6-1-1

Table 6-1-2

[0203] This embodiment describes the production of a GAA variant. GAA production in EXPI293F(trademark) cells

[0204] Milligram-scale production of GAA variants was achieved by transient transfection of EXPI293® cells (ThermoFisher Scientific) using a lipofection method with EXPIFECTAMINE® 293 Reagent (ThermoFisher Scientific) in EXPI293® Expression Medium (ThermoFisher Scientific). GAA variants fused to an N-terminal synthetic mammalian signal peptide (SEQ ID NO: 3381) were subcloned into the mammalian expression vector pLEV113, pcDNA 3.1(+), or pDH as described in Example 1. EXPI293® cells were transfected with plasmid DNA and grown in suspension for 4–7 days. Conditional media were then collected, clarified by centrifugation and filtration, and stored at 4°C until analysis. (Example 8) Purification of GAA variants

[0205] This embodiment describes a method for purifying the GAA variant. Purification of GAA variants from mammalian cell supernatant

[0206] As described in Example 7, GAA variants (SEQ ID NOs: 4, 6, 10, 12, 16, 18, 20, 946, 1894, 1924, 1950, 1956, 1984, 2034, 2054, 2066, 2074, 2178, 2202, and 2496) produced in EXPI293F® cells were purified from mammalian culture supernatant as described in the literature (Yasuda et al., Prot. Exp. Pur, 37:499-506

[2004] ). 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 sterile filtered through a 0.2 μm bottle-top filter and then loaded onto a column. After loading, the column was washed with 10 column volumes of concanavalin A-binding buffer, and the bound proteins were 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 proteins were concentrated and the buffer was changed to a 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 equipped with a 50 kDa membrane (Millipore). The GAA in the storage buffer was filtered through an ANOTOP® 0.2 μm syringe filter (Whatman) for sterile filtration, and the buffer was stored at -80°C. Based on BCA quantification (described below), this purification process yielded 2.4–50 ng of purified protein per 1 ml of culture supernatant. Protein quantification by BCA assay

[0207] The purified GAA variants produced as described above were quantified using the Bicinchoninic acid (BCA) protein assay (Sigma Aldrich). In microtiter plates, 25 μL of protein standards and appropriately diluted purified GAA were mixed with 200 μL of working reagent containing 50 parts of BCA reagent A and 1 part of BCA reagent B. The plates were carefully mixed on a plate shaker for 30 seconds and incubated at 37 °C for 30 minutes. Absorbance was measured at 562 nm using a plate reader. (Example 9) In vitro Characterization of GAA Variants

[0208] In this example, the experiments conducted to characterize the GAA variants produced as shown herein are described. Kinetic Characterization of rhGAA and GAA Variants

[0209] The activity of the GAA variants was determined by measuring the hydrolysis of 4-methylumbelliferyl α-D-glucopyranoside (4-MUGlu). The purified enzyme produced as described above was mixed with 50 μL of 0 - 2.5 mM 4-MUGlu in McIlvaine buffer (McIlvaine, J. Biol. Chem., 49:183 - 186

[1921] ), pH 4.5, in a 96-well, black, opaque-bottom plate. The reaction was mixed briefly and incubated at 37 °C for 15 - 30 minutes, after which the reaction was stopped with 100 μL of 0.5 M sodium carbonate, pH 10.5. Hydrolysis was analyzed by monitoring fluorescence (Ex. 355 nm, Em. 460 nm) using a SPECTRAMAX® M2 microplate reader, plotted, and analyzed using the Michaelis-Menten equation. The results from this assay are presented in Figure 1. Stability of rhGAA and GAA Variants at Neutral pH

[0210] The stability of the GAA variant to neutral pH was determined by incubating the variant in 100 μL of MEM complete growth medium (pH 7.4) in a 96-well plate. The plate was sealed and incubated at 37°C for up to 48 hours. Next, 10 μL of the neutral pH exposed sample was mixed with 50 μL of 1.5 mM 4-MUGlu in McIlbine buffer, pH 4.4, in a 96-well, black, opaque-bottom plate. After incubation at room temperature for 30 minutes with agitation, the reaction was stopped with 100 μL of 0.5 M sodium carbonate, pH 10.5. Hydrolysis of 4-methylumbelliferyl α-D-glucopyranoside (4-MUGlu) was analyzed by monitoring fluorescence (Ex. 355 nm, Em. 460 nm) using a SPECTRAMAX® M2 microplate reader or an ENVISION® microplate reader (Perkin Elmer). The results from this assay are shown in Figure 11. Cellular uptake of purified GAA variant expressed in Expi293F cells into Pompe fibroblasts or C2C12 GAA knockout myoblasts.

[0211] The cellular uptake of GAA variants was determined by comparing them with reference enzymes (SEQ ID NOs: 4 and 6) to assess their overall ability to be endocytosed in cultured cells. Pompe fibroblasts (GM00248, Coriell Institute for Medical Research) or C2C12 GAA knockout cells were seeded in standard complete growth medium in black-walled, clear-bottomed 96-well plates (Costar, #3603) and allowed to reach confluence (2-3 days at 37°C, 5% CO2). After confluence, the standard complete growth medium was removed using an automated BIOMEK® i5 liquid processing robot. The purified enzyme, as described in Example 8, was added to the cells in standard complete growth medium at a concentration of 0-10 ug GAA / mL and incubated at 37°C, 5% CO2 for acute (1-6 hours) or long-term (16-96 hours) treatment. The medium containing the GAA variants was aspirated using an automated BIOMEK® i5 liquid processing robot. Cells were briefly washed with 150 μL of 1× DPBS / well, and the DPBS was removed using an automated BIOMEK® i5 liquid processing robot. Then, 200 μL of standard complete growth medium was added to each well, and the plates were returned to the incubator for 0–72 hours. Upon completion of incubation, the MEM complete growth medium was removed using an automated BIOMEK® i5 liquid processing robot. Cells were washed with 150 μL of 1× DPBS / well, and the DPBS was removed using an automated BIOMEK® i5 liquid processing robot. Cells were lysed by adding 50 μL of McIlbine buffer, pH 4.4, supplemented with 0.2–0.5% TRITON X-100® nonionic surfactant (Sigma #93443), and stirring at room temperature for 30 minutes. GAA activity was assessed by adding 50 μL of 1.5 mM 4-MUGlu in McIlbine buffer, pH 4.4. The plates were sealed and incubated at 37°C for 300–360 minutes with stirring at 400 rpm, after which the reaction was stopped with 100 μL of 0.5 M sodium carbonate, pH 10.5. Hydrolysis was analyzed by monitoring fluorescence (Ex. 355 nm, Em. 460 nm) using a SPECTRAMAX® M2 microplate reader.FIOPC for cellular uptake was calculated by dividing the intracellular activity of the normalized GAA variant by the control (WT) activity. Figures 4, 5, 8, 12, 13, 16-18, 22, 24, and 26 provide graphs showing the cellular uptake of purified GAA variants into Pompe fibroblasts after 1-96 hours of treatment. Figures 6, 7, 14, 15, 19-21, 23, 25, and 27 provide graphs showing the cellular uptake of purified GAA variants into C2C12 GAA KO myoblasts after 1-96 hours of treatment. Figure 8 provides graphs showing the cellular uptake and stability of GAA variants after 1-7 days. Glycogenolytic activity of GAA variant expressed in EXPI293F(trademark) cells

[0212] The relative glycogen hydrolysis activity of GAA variants was assessed using the AMPLEX® Red Glucose / Glucose Oxidase Kit (Invitrogen, #A22189) according to the manufacturer's instructions for use, with the modifications described herein. Briefly, the GAA variants were diluted with 10 mM glycogen in McIlvine buffer, pH 4.4, to a concentration range (0–2 μg / mL) appropriate for a 50 μL reaction in a COSTAR® black-walled, clear-bottomed 96-well plate (#3603, Corning). The plate was sealed and gently shaken at room temperature for 30 minutes. The reaction mixture was neutralized by adding 25 μL of 0.5 M sodium carbonate, pH 10.5. 50 μL of AMPLEX® Red / horseradish peroxidase / glucose oxidase solution, prepared according to the manufacturer's instructions for use, was added to each well. The plate was sealed and gently shaken at room temperature for 30 minutes. Using a SPECTRAMAX® M2 microplate reader, the reactants were quantified against a glucose-free standard curve by monitoring fluorescence (Ex. 530 nm, Em. 590 nm). The results are shown in Figure 2. (Example 10) GAA variant of Sequence ID No. 20

[0213] This example describes experiments on the evolution of GAA variants derived from Sequence ID No. 20, and screening for improvement in GAA activity after a series of exposures. A library of variant genes GAA encoded by / based on Sequence ID No. 20 was constructed, seeded, and grown as described in Example 5, and screened for GAA MU-Glu activity ("FIOPC of unexposed activity") and also after plasma exposure ("FIOPC of plasma stability and activity"). The variants were also screened for 4-MuGlu activity after lysing of Pompe fibroblasts ("FIOPC of activity from Pompe fibroblast lysates") or GAA as described in Example 5. - / - After lysing of C2C12 cells ("C2C12 GAA") - / - The activity of the lysates was tested against FIOPC. The results of these assays are presented in Tables 10-1 and 10-2. [Table 10-1-1] [Table 10-1-2] [Table 10-1-3] [Table 10-1-4] [Table 10-1-5] [Table 10-2-1] [Table 10-2-2] [Table 10-2-3] [Table 10-2-4] [Table 10-2-5]

Table 10-2-6

Table 10-2-7

Table 10-2-8

Table 10-2-9

Table 10-2-10

[0214] pDH or PCR-amplified linear DNA (as described in Example 1) encoding synthetic mouse IG signal peptides (SEQ ID NOs: 3381 and 3382) fused to wild-type GAA or GAA variants was transfected into EXPI293F™ cells (ThermoFisher Scientific) using a lipofection method 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 seeded at 1×10 6Cells were seeded at a density of 400 μL / well. Cells were subjected to lipofection-mediated transfection and returned to a shaking incubator with 8% CO2 and 70% humidity for 3–4 days to allow expression of the GAA variant and secretion into the conditional medium. The conditional medium was collected by centrifugation of the expression plate and transfer of the conditional medium to a BioRad Hardshell PCR Plate (BioRad, HSP9601). The plate was centrifuged again, and the clarified conditional medium was transferred to a new 96-well plate for analysis of activity, stability, or uptake into cells. HTP analysis of the supernatant

[0215] GAA variant activity was determined by measuring the hydrolysis of 4-methylumbelliferyl α-D-glucopyranoside (4-MUGlu). For non-exposure assays, 5–10 μL of EXPI293F™ clarified medium, prepared as described above, was mixed with 50 μL of 1.5 mM 4-MUGlu in McIlvaine buffer (McIlvaine, J. Biol. Chem., 49:183-186

[1921] ), pH 4.4, in a 96-well, black, opaque-bottom plate. The reaction was incubated at 25–37°C for 10–60 minutes with stirring at 400 rpm, and then stopped with 100 μL of 0.5 M sodium carbonate, pH 10.5. Hydrolysis was analyzed by monitoring fluorescence (Ex. 355 nm, Em. 460 nm) using a SPECTRAMAX® M2 microplate reader (Molecular Devices) or an ENVISION® microplate reader (Perkin Elmer). Unexposed activity FIOPC was calculated by dividing the normalized GAA variant by the activity of the reference polypeptide having the indicated SEQ ID NO. HTP analysis of supernatant exposed to plasma

[0216] GAA variants were exposed to plasma to simulate the conditions they would encounter in the blood after administration to a patient. First, 30 μL of GAA variant in EXPI293F® clarified 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 2–4 hours. Next, 10 μL of the plasma-exposed sample was mixed with 50 μL of 1.5 mM 4-MUGlu in McIlbine buffer, pH 4.4. The reaction was incubated at 25–37°C for 15–60 minutes with stirring at 400 rpm, and then the reaction was stopped with 100 μL of 0.5 M sodium carbonate, pH 10.5. Hydrolysis was analyzed by monitoring fluorescence (Ex. 355 nm, Em. 460 nm) using a SPECTRAMAX® M2 microplate reader or an ENVISION® microplate reader (Perkin Elmer). Plasma stabilization FIOPC was calculated by dividing the normalized GAA variant activity after exposure by the activity of the reference polypeptide having the indicated sequence number. HTP analysis of GAA activity in lysates of Pompe fibroblasts and C2C12 GAA knockout myoblasts

[0217] GAA variants derived from HTP EXPI293F® expression in clarified medium were incubated with target cells, and residual intracellular activity was assayed after 24–72 hours. These experiments used mammalian cells lacking functional GAA activity, namely Pompe patient-derived fibroblasts (Coriell Institute for Medical Research #GM00248) and C2C12 myoblasts in which the native GAA gene was knocked out using Crispr-Cas9 editing. In these experiments, Pompe fibroblasts or C2C12 GAA knockout myoblasts were seeded in 96-well COSTAR® plates (3904, Corning) and grown in standard complete growth medium until confluence. Once confluence was reached, the complete growth medium was removed from the plates using an automated BIOMEK® i5 liquid handling robot. Clarified media from transient HPT transfection of EXPI293F® were transferred to Pompe patient-derived fibroblasts and C2C12 myoblasts and incubated for 4–24 hours at 37°C and 5% CO2. The medium was removed from the culture using an automated BIOMEK® i5 liquid processing robot. The cells were briefly washed with 150 μL of 1× DPBS / well, and the DPBS was removed using an automated BIOMEK® i5 liquid processing robot. Then, 200 μL of standard complete growth medium was adde...

Claims

1. Recombinant acid alpha-glucosidase comprising an amino acid sequence having at least 97%, at least 98%, or at least 99% sequence identity with respect to the sequence of residues 20-944 of SEQ ID NO: 3116, wherein the amino acid sequence, compared to the amino acid sequence of SEQ ID NO: 2, has the following substitutions at the following positions: L24W, L28S, L29T, P39Q, Q50V, A62L, P78E, L109D, S135Q, T150S, A recombinant acid alpha-glucosidase comprising T266N, R267K, A437G, T486E, E522V, L569T, T692G, A711H, A750P, A812E, Q830K, L860F, L871E, R883H, Q894G, V913R, and S932A, wherein the recombinant acid alpha-glucosidase exhibits increased tolerance to pH 7 compared to the acid alpha-glucosidase of SEQ ID NO:

2.

2. Recombinant acid alpha-glucosidase according to claim 1, derived from human acid alpha-glucosidase.

3. The recombinant acid alpha-glucosidase according to claim 1 or 2, further exhibiting, compared to SEQ ID NO: 2, at least one improved property selected from any combination of i) enhanced catalytic activity, ii) increased tolerance to pH 4, iii) increased expression, iv) increased uptake into cells, v) increased enzyme activity in cell lysates, vi) decreased immunogenicity, or i) ii) iii) iv) v) and / or vi).

4. A composition comprising at least one recombinant acid alpha-glucosidase according to any one of claims 1 to 3.

5. A recombinant polynucleotide encoding at least one recombinant acid alpha-glucosidase according to any one of claims 1 to 4.

6. Recombinant polynucleotide according to claim 5, selected from DNA, RNA, and mRNA, and optionally codon-optimized.

7. An expression vector comprising a recombinant polynucleotide according to claim 5 and / or 6, wherein the recombinant polynucleotide is optionally operably linked to a control sequence, the control sequence is optionally a promoter, and the promoter is optionally a heterologous promoter.

8. A host cell comprising the expression vector described in claim 7, wherein the host cell is optionally selected from eukaryotes and prokaryotes, and is optionally a mammalian cell.

9. A method for producing a recombinant acid alpha-glucosidase variant, comprising the steps of culturing the host cells described in claim 8 under conditions that produce the acid alpha-glucosidase encoded by the recombinant polynucleotide, optionally further comprising the step of recovering the acid alpha-glucosidase, and optionally further comprising the step of purifying the acid alpha-glucosidase.

10. A recombinant acidic alpha-glucosidase variant produced according to the method described in claim 9.

11. A composition comprising the recombinant acid alpha-glucosidase described in claim 10.

12. A composition comprising at least one polynucleotide according to claim 5 or claim 6.

13. A pharmaceutical composition for the treatment of Pompe disease, comprising the composition according to claim 4, 11, or 12, wherein the pharmaceutical composition is optionally suitable for parenteral injection or infusion into humans.

14. A pharmaceutical composition comprising a recombinant polynucleotide according to claim 5 or claim 6, wherein the pharmaceutical composition is optionally suitable for parenteral injection or infusion into humans.

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

16. A pharmaceutical composition and / or composition according to any one of claims 4, 11, 12, 13, 14 and / or 15 for treating and / or preventing symptoms of Pompe disease in a subject.

17. A pharmaceutical composition according to claim 16 and / or a composition which improves the symptoms of Pompe disease, wherein the subject is optionally an infant or a child, or optionally an adult or a young adult.

18. Use of the composition provided in any of claims 4 and 11 to 15 in the manufacture of a medicament for treating and / or preventing symptoms of Pompe disease in a subject.