Engineered acid alpha-glucosidase variants

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Engineered acidic alpha-glucosidase polypeptides with improved stability and activity are developed to enhance enzyme replacement therapy for Pompe disease, addressing the limitations of current treatments.

JP2025089465AInactive Publication Date: 2025-06-12CODEXIS INC

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Patent Information

Application Number: JP2025050060
Authority / Receiving Office: JP · JP
Patent Type: Applications
Current Assignee / Owner
Priority Date: 2019-12-20
Filing Date: 2025-03-25
Publication Date: 2025-06-12
Estimated Expiration: Not applicable · inactive patent

Application Information

Patent Timeline

20 Dec 2019

Priority

25 Mar 2025

Application

JP2025050060

12 Jun 2025

Publication

JP2025089465A

IPC: C12N9/26; C12N15/56; C12N15/63; C12N15/113; C12N5/10; C12N1/21; C12N1/19; C12N1/15; C12P21/02; A61K38/47; A61K48/00; A61P21/04

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

Inventors: ウィリアムケイシーハロウズ; レイチェルキャスリーンボサム

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AI Technical Summary

Technical Problem

Current treatments for Pompe disease, particularly enzyme replacement therapy, are inadequate in providing sustained enzyme activity and effective management of the disease, especially in severe infantile forms.

Method used

Development of engineered acidic alpha-glucosidase (GAA) polypeptides with enhanced expression, stability at neutral and acidic pH, cellular uptake, and activity in cell lysates, which are used in therapeutic compositions to address Pompe disease.

Benefits of technology

The engineered GAA polypeptides demonstrate improved stability, activity, and cellular uptake, potentially leading to more effective enzyme replacement therapy and better management of Pompe disease.

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Abstract

To provide engineered acid alpha-glucosidase variants.SOLUTION: The invention provides engineered acid alpha-glucosidase (GAA) polypeptides and compositions thereof. In some embodiments, the engineered GAA polypeptides have been optimized to provide increased expression, increased stability at neutral pH, and increased activity in cell lysates. The invention also provides methods for utilizing the compositions comprising the engineered GAA polypeptides for therapeutic or other purposes.SELECTED DRAWING: Figure 1

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Description

Technical Field

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

[0002] Field of the Invention The present invention provides engineered acidic 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 cellular uptake, and increased activity in cell lysates. The present invention also provides methods for using compositions comprising the engineered GAA polypeptides for therapeutic and other purposes.

[0003] Reference to a Sequence Listing, Table, or Computer Program A formal copy of the Sequence Listing is being filed herewith as an ASCII text file named "CX7-199WO2_ST25.txt", created on December 17, 2020, and having a size of 18.2 megabytes, via EFS-Web simultaneously with this specification. This Sequence Listing filed via EFS-Web is part of this specification and is hereby incorporated by reference in its entirety.

Background Art

[0004] Background of the Invention Pompe disease is an autosomal recessive lysosomal storage disorder caused by mutations 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 within lysosomes can lead to lysosomal swelling and rupture, which can in turn result in cellular injury, organelle dysfunction, and other cellular defects. There are two types of Pompe disease, including the classic infantile and late-onset (childhood or adult) forms. The disease severity is related to the amount of enzyme activity present in the cells of affected individuals. The infantile form is the most severe and rapidly progressive type, usually with less than 1% GAA activity, resulting in significant glycogen accumulation not only in skeletal muscle but also in the heart and other tissues (see, for example, Hahn and Schanzer, Ann. Transl. Med., 7:283

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

[2019] ). Patients It presents with elevated creatine kinase levels, hypertrophic cardiomyopathy, growth impairment, muscle hypotonia, and reduced axial muscle strength. Without treatment, patients typically die within the first year of life due to cardiorespiratory failure. Survival beyond 18 months of age is exceptional. This type is distinguished from the non-classical or late-onset infantile form of Pompe disease, in which patients present with a much less severe form of cardiac hypertrophy. Patients with late-onset Pompe disease generally experience progressive limb-girdle myopathy and respiratory dysfunction. These patients present with a dominant myopathy, although not exclusively. Patients will ultimately become wheelchair and / or ventilator dependent. Respiratory failure is the main cause of death in these patients. There are patients who can synthesize non-functional forms of GAA, and there are also patients who cannot produce any type of native enzyme. The human GAA gene encoding GAA has been identified as located at 17q25.2-q25.3 and has been cloned and sequenced (see Peruzzo et all, Ann. Transl. Med., 7:278-287

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

[1991] ). Although a very large number of mutations in this gene have been reported, the pathological mechanisms leading to the wide range of phenotypes observed in affected patients remain unclear. Despite the availability of enzyme replacement therapy (ERT) with recombinant GAA, better treatment and management options for affected patients are still needed.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

[2019]

Non-Patent Document 2

[2019]

Non-Patent Document 3

[2019]

Non-Patent Document 4

[1991]

Summary of the Invention

Means for Solving the Problems

[0006] Summary of the Invention The present invention provides engineered acidic 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 cellular uptake, and increased activity in cell lysates. The present invention also provides methods for using 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") and their bioactive fragments and analogs that have improved properties when compared to wild-type GAA enzyme and / or reference GAA polypeptides under essentially the same conditions. The present invention further relates to methods of using engineered GAA polypeptides and their bioactive fragments and analogs in therapeutic and / or other compositions.

[0007] The present invention provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104. In some embodiments, the present invention provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104. In some embodiments, the present invention further provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment comprising an amino acid sequence comprising SEQ ID NO: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104. In some embodiments, the present invention further provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment comprising an amino acid sequence consisting of SEQ ID NO: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104. In some embodiments, the recombinant acidic alpha-glucosidase sequence comprises a signal peptide sequence (e.g., SEQ ID NO: 3382 or 3384) encoded by the polynucleotides set forth in SEQ ID NO: 3381 and 3383, respectively.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 comprising the recombinant acid alpha-glucosidase does not include a signal peptide. It is not intended that the present invention be limited to recombinant acid alpha-glucosidase polynucleotide or polypeptide sequences that include a signal peptide nucleotide or polypeptide sequence. It is also not intended that the present invention be limited to recombinant acid alpha-glucosidase polynucleotide or polypeptide sequences that do not include a signal peptide nucleotide or polypeptide sequence.

[0008] The present invention provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO:2. In some embodiments, the acidic alpha-glucosidase comprises at least one substitution at a position or set of positions selected from 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, 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, these positions being numbered with reference to SEQ ID NO:2. In some embodiments, the acidic alpha-glucosidase is 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, 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 includes at least one substitution or set of substitutions at one or more positions selected from F27P, F27P / C944W, F27R, L28P, L28R, L28S, L29T / A478T, V30G, V30K, V30T, K88G, K88S, Q107G, Q107P, L109G / G842E, L109P, Q110G, Q110L, Q113S, S135A, S135Q, E137P, M138A, T148G, T148Y, T150G, Q247R, D274G, A276F, A276Y, T278A, T278G, I375E, R403W, R414P, A418E / H499R, A418R, Q421S, G426R, A437S, A444T, R455V, 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, and these positions are numbered with reference to SEQ ID NO: 2. In some embodiments, the acid alpha-glucosidase includes at least one substitution or set of substitutions at one or more positions selected from 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、At least one substitution is included at a position or set of positions selected from 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, 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, and these positions areNumbered with reference to SEQ ID NO: 2. In some embodiments, the 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 / 894R106P / 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 / 793G180H / 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 / 671N275V / 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, 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 at least one substitution or set of substitutions at one or more positions selected from 547G / 638I / 668D, wherein these positions are numbered with reference to SEQ 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 / Q894RK106P / 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 / S668DL275M / 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 / S671NOne or more substitutions or sets of substitutions at one or more positions selected from 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 A547G / V638I / S668D, these positions being numbered with reference to SEQ ID NO: 2. In some embodiments, the 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、Comprising at least one substitution or set of substitutions at one or more positions selected from 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, 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 / 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, these positions being numbered with reference to SEQ ID NO: 2. In some embodiments, the 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 / 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 / 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 / 671N275M / 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, comprising at least one substitution or set of substitutions at one or more positions selected from 8V / 610R, 402A / 431V / 547G / 638I / 671N, 403W, 414P, 418E / 499R, 431V / 507L / 518V / 541E / 547G / 638I / 669H / 671N, 431V / 507L / 518V / 669H / 671N, 437S, 471Q / 478S, 507L / 547G / 610R, 507L / 547G / 638I / 669H / 671N, 547G, 547G / 610R / 638I / 671N, 547G / 638I / 668D, 581G, 581T, 642Q, 642S, 670N, 692Q, 750P, 753T, 820E, 871E, and 944G, these positions being numbered with reference to SEQ 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 / I744V / I869T, L29T / A478T, L29V / L218S / L240I / H700F / I869T, V30G, V30K, V30T, 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 / 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 / E793KK106P / 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 / S668DT269N / 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 / 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,One 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 a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO: 6. In some embodiments, the acidic 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 / 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、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、At least one substitution is included at a position or set of positions selected from 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, 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 / 547 / 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, and these positions are numbered with reference to SEQ ID NO: 6. In some embodiments, the 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 / 894R106P / 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 / 671N191R / 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 / 715G275V / 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, 610AContains at least one substitution at 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, and these positions are numbered with reference to SEQ 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 / L240I / H700F / I869T, V30G, V30K, V30T, G36R / K106P / T150S / L218S / N527D / A750P / R883H / Q894R, K88G, K88S, 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 / Q894RK106P / 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 / S671NL275V / 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,Comprising at least one substitution at a position or set of positions selected from 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, A547G / 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, these positions being numbered with reference to SEQ ID NO: 6.,

[0010] The present invention provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO:8. In some embodiments, the acidic alpha-glucosidase comprises at least one substitution at a position or set of positions selected from 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 / 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, these positions being numbered with reference to SEQ ID NO:8. In some embodiments,Acid alpha-glucosidase contains at least one substitution or set of substitutions at one or more positions selected from 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 / 749E, 106P / 218S / 281V, 106P / 218S / 455V, 106P / 218S / 455V / 507L / 749E, 106P / 489R / 671N, 106P / 638I, 106P / 671N / 934R, 113S, 113S / 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 / 281V, 218S / 281V / 431V, 218S / 281V / 671N, 218S / 431V, 218S / 431V / 489R / 507L / 749E / 934R, 218S / 455V, 218S / 507L / 749E, 218S / 507L / 934R, 218S / 638I / 671N, 218S / 749E, 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, and these positions are numbered with reference to SEQ ID NO:8. In some embodiments, acid 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, 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 / L934Rand contains at least one substitution or set of substitutions at one or more positions selected from Q749E / A784, these positions being numbered with reference to SEQ ID NO: 8. In some embodiments, the acid alpha-glucosidase contains at least one substitution or set of substitutions at one or more positions selected from 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 / 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, these positions being numbered with reference to SEQ ID NO: 8. In some embodiments, the acid alpha-glucosidase contains at least one substitution or set of substitutions at one or more positions selected from 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 / 581T, 135P / 944Y, 135Q, 135Q / 148G / 150G / 418E, 135Q / 478T / 489R / 581T, 138A / 218S / 668D / 671N, 157M, 218S / 638I / 671N, 418E, 418E / 489R,comprises at least one substitution or set of substitutions at one or more positions selected from 431V / 668D / 671N, 507L / 668D, and 671N / 749E, these positions being numbered with reference to SEQ ID NO:8. In some embodiments, the acid alpha-glucosidase comprises at least one substitution or set of substitutions at one or more positions selected from 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 / A489R / S671N, K106P / V638I, Q113S / S135Q / A418E, Q113S / A418E / R455V / A478T / K581T, Q113S / A418E / A478T / A489R / K581T, S135P / C944Y, S135Q, S135Q / T148G / S150G / A418E, S135Q / A478T / A489R / K581T, M138A / L218S / S668D / S671N, L157M, L218S / V638I / S671N, A418E, A418E / A489R, M431V / S668D / S671N, M507L / S668D, and S671N / Q749E, these positions being numbered with reference to SEQ ID NO:8.,

[0011] The present invention also provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO: 12. In some embodiments, the acidic alpha-glucosidase comprises at least one substitution at a position or set of positions selected from 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 / 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, and these positions are numbered with reference to SEQ ID NO: 12. In some embodiments, the acidic alpha-glucosidase comprises 4H, 27P / 28S / 489R, 27P / 418E / 478T, 27R,At least one substitution is included at a position or set of positions selected from 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 / 218S / 281V, 106P / 218S / 455V, 106P / 218S / 455V / 507L / 749E, 106P / 489R / 671N, 106P / 638I, 106P / 671N / 934R, 113S, 113S / 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 / 281V / 431V, 218S / 281V / 671N, 218S / 431V, 218S / 455V, 218S / 507L / 749E, 218S / 638I / 671N, 218S / 749E, 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, and these positions are numbered with reference to SEQ ID NO: 12. In some embodiments, the acid 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,Comprising at least one substitution at a position or set of positions selected from 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 / M431V, L218S / A281V / S671N, L218S / M431V, L218S / R455V, L218S / M507L / Q749E, 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 Q749E / A784T, these positions being numbered with reference to SEQ ID NO: 12.,

[0012] The present invention provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO: 14. The present invention provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO: 16. In some embodiments, the acidic 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 / 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, 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 / 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,comprising at least one substitution at a position or set of positions selected from 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, 448, 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, wherein these positions are numbered with reference to SEQ ID NO: 14 and / or 16. In some embodiments, the 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 / 793K, 37F / 196T, 37F / 528S / 696S / 793K, 37F / 528S / 790V, 37F / 528S / 790V / 793K / 862Q, 37F / 790V / 793K, 39D, 39H, 39Q, 39Q / 58L / 489D / 725E / 830K / 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, 944Rcomprising at least one substitution or set of substitutions at one or more positions selected from 944S, these positions being numbered with reference to SEQ ID NO: 14 and / or 16. 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 / K154R / D523N / E681Q / E793K / R862Q, S37F / N79S / K154R / E793K, S37F / A196T, S37F / N528S / A696S / E793K, S37F / N528S / I790V, S37F / N528S / I790V / 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 / Q830K / 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, A62E / N79S / R862Q, A62E / Q92R, A62E / Q92R / I790V / E793K, A62E / K106R / D523N / N528S / A696S / E793K / R862Q,One or more substitutions or sets of substitutions at one or more positions selected from 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 / L860F / C930P / C944S, V70Q, P77W, N79S / K154R / E681Q, N79S / K154R / E793K / R862Q, N79S / R862Q, A89R, A97D, A97G, K106R / 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, E208G, E208I, Q217A, Q217D, R267K / A489D / D500A / K725E / Q830K / C930P, R267K / E522V / K725E, Y352K, Y352V, R385G, H424K, R448L, E463A, A489D / Q830K / C944S, D500A, D500A / S612D / Q830K / L860F, D500A / L860F / C930P, D500A / C930P / C944S, E522V / K725E, D523N, D523N / I790V / E793K, N528S / E681Q, N528S / E793K, N528S / R862Q, L672E, L672K, P673N, P673R, K725F, 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, wherein these positions are numbered with reference to SEQ ID NO: 14 and / or 16.,

[0013] The present invention also provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO: 18, wherein the positions are numbered with reference to SEQ ID NO: 18. In some embodiments, the acidic alpha-glucosidase comprises at least one substitution at a position or set of positions selected from 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, 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, and these positions are numbered with reference to SEQ ID NO: 18.In some embodiments, the acid alpha-glucosidase comprises at least one substitution at a position or set of positions selected from 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, 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, and these positions are numbered with reference to SEQ ID NO: 18.In some embodiments, the acid alpha-glucosidase comprises at least one substitution at a position or set of positions selected from 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 / D523N, S37F / A196T, S37F / N528S / I790V, P39D, P39Q, P39Q / V70A / L109P / Q830K / G842S, P39Q / V70A / K725E, 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 / E522V / K725E, Y352K, Y352V, R385G, D500A / C930P / C944S, P673N, H734K, A774G, L778Q, N875D, C930P, S932A, and L934F, and these positions are numbered with reference to SEQ ID NO: 18.

[0014] The present invention also provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to the amino acid sequence numbered with reference to SEQ ID NO: 20. In some embodiments, the acidic 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 / 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,At least one substitution is included at a position or set of positions selected from 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, 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, and these positions are numbered with reference to SEQ 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,Comprising at least one substitution at a position or set of positions selected from 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, 57M, 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, 108R, 109D / 612S / 774S / 944S, 112H, 123L, 123V, 124G, 124M, 124V, 148K, 148R, 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, 727W, 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, and these positions are numbered with reference to SEQ ID NO: 20. In some embodiments,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,At least one substitution in a position or set of positions selected from 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, A89R / A97G / Q107G, A89R / A97G / D500A, A89R / S842G, Q107G / L109D, Q107G / D500A / 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, S402N, S402V / P781Q, D489A, D489A / C944R, D500A / S842G, D500A / S932A, D523E, N527R, N527V, D612S, D612S / K725E / C944S, D612S / P922E, E614Q, E614R, E614W, S727W, L742V, L748V, G820A, G820V, L823F, L823V, P832A, P832R, 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, these positions being numbered with reference to SEQ 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、 , at least one substitution is included at a position or set of positions selected from 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, 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, 264, 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, and these positions are numbered with reference to SEQ 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, 108Hat least one substitution at a position or set of positions selected from 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, 253G, 253P, 259G, 259N, 259S, 260W, 261E, 262P, 264C, 279E, 305F, 305G, 305R, 305V, 305Y, 309C, 309G, 312A, 319F, 320M, 329F, 333L, 333V, 387L, 402G, 402N, 421P, 432C, 500A / 842G, 500A / 932A, 556H, 556R, 556S, 556Y, 612S, 612S / 725E / 944S, 612S / 922E, 727G, 727Q, 727T, 727W, 736M, 736V, 736W, 737M, 741C, 741D, 741E, 741G, 741T, 742V, 748I, 748T, 748V, 815A, 815M, 816V, 818T, 818V, 823A, 823F, 823G, 823R, 832E, 832G, 842G / 932A, 911G, 913A, 913E, 913G, 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, these positions being numbered with reference to SEQ ID NO: 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, K106Ncomprising at least one substitution at a position or set of positions selected from K106T, Q107G / L109D, Q107G / D500A / S842G, G108H, G108N, G108R, G108S, G108V, L109D / D612S / A774S / C944S, A112H, A112P, T148E, T148G, T148H, 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, L259S, M260W, L261E, S262P, S264C, P279E, L305F, L305G, L305R, L305V, L305Y, 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, S727Q, S727T, S727W, L736M, L736V, L736W, L737M, A741C, A741D, A741E, A741G, A741T, L742V, L748I, L748T, L748V, I815A, I815M, I816V, L818T, L818V, L823A, L823F, L823G, L823R, P832E, P832G, S842G / S932A, N911G, 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, these positions being numbered with reference to SEQ ID NO: 20.,

[0015] The present invention further provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO: 946, wherein the positions are numbered with reference to SEQ ID NO: 946. In some embodiments, the acidic 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 / 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 / 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,At least one substitution is included at a position or set of positions selected from 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 / 614 / 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 / 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, and these positions are numbered with reference to SEQ ID NO: 946. In some embodiments, the 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 / 500A / 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, 57F / 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 / 858W, 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 / 932AAt least one substitution is included at a position or set of positions selected from 62L / 437G / 913R / 916R, 62L / 489A / 500A / 932A, 62L / 489A / 614Q / 916R, 62L / 527R, 62L / 527R / 727W / 916G, 62W / 89R / 124V / 381W / 858C, 62W / 89R / 149R / 381W, 62W / 89R / 188W / 489A / 500A / 727W / 823F, 62W / 89R / 381V, 62W / 89R / 381W / 858C, 62W / 124V / 148R / 149R / 381W / 614R, 62W / 124V / 149R / 381V / 832R / 858C / 937K, 62W / 124V / 188W / 823F / 842G / 862Q, 62W / 148R / 149R / 381V / 858C / 937K, 62W / 148R / 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, 124V / 148R / 381W / 727W / 858W / 937K, 124V / 381W / 614R, 124V / 500A / 842G / 862Q, 124V / 832R / 937K, 148R / 832R / 858W / 937K, 381V, 381V / 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, and these positions are numbered with reference to SEQ ID NO: 946. In some embodiments, the acid 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 / D923WA62F / 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 / S932A, A62L / A437G / N527R / S727W, A62L / A437G / N527R / S916G / S932A, A62L / A437G / 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 / E381W / 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 / E381W / E858C, A62W / P149R / E381W / E614R / E937K, A62W / E381V, A62W / A437G / D489A / E614Q / S727W / V913R, A62W / D489A / N527R / S916R / S932A, A62W / D500A, A62W / S727W, A62W / S916G, A89R / T148R / P149R, A89R / T148R / P149R / E381W, A89R / E381W, S124V / T148R / E381W / S727W / E858W / E937K, S124V / E381W / E614R, S124V / D500A / S842G / R862Q, S124V / P832R / E937K, T148R / P832R / E858W / E937K, E381V, E381V / E614R / P832R, E381W / E858C / E937K, A437G, A437G / D489A / P914R / S916R, A437G / S727W / P914K, A437G / P914R / S916G, D489A / D500AContains at least one substitution at a position or set of positions selected from D489A / E614Q / S916G, D500A / S727W / V913R / S916R, D500A / P914R / S916G, and D923W, and these positions are numbered with reference to SEQ ID NO: 946. In some embodiments, the acidic 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 / 914, 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 / 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 contains at least one substitution at a position or set of positions selected from 923, and these positions areNumbered with reference to Sequence Number 946. In some embodiments, the acid alpha-glucosidase is 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 / 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 / 188W / 489A / 823F / 862Q, 62L / 305F / 437G / 500A / 727W / 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 / 727W / 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,Contains at least one substitution at a position or set of positions selected from 489A / 614Q / 916G, 500A / 727W / 913R / 916R, 500A / 914R / 916G, and 923W, and these positions are numbered with reference to SEQ ID NO: 946. In some embodiments, the acid 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 / 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 / E188W / D489A / L823F / R862Q, A62L / L305F / A437G / D500A / S727W / V913R, A62L / L305F / S727W, A62L / A437G / D489A / N527R / S727W / V913R / S932A, A62L / A437G / N527R / S727W, A62L / A437G / N527R / S916G / S932A, A62L / A437G / V913R / S916R,Comprising at least one substitution at a position or set of positions selected from A62L / D489A / D500A / S932A, A62L / N527R / S727W / S916G, A62W / A89R / E188W / D489A / D500A / S727W / L823F, A62W / S124V / E188W / L823F / S842G / R862Q, A62W / A437G / D489A / E614Q / S727W / V913R, A62W / D489A / N527R / S916R / S932A, A62W / S727W, A62W / S916G, S124V / D500A / 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, these positions being numbered with reference to SEQ ID NO: 946.,

[0016] The present invention provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO: 1956, wherein the positions are numbered with reference to SEQ ID NO: 1956. In some embodiments, the acidic 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 / 536 / 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,and includes at least one substitution at a position or set of positions selected from 909, these positions being numbered with reference to SEQ ID NO: 1956. In some embodiments, the acid 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, 145R, 154R / 588L, 156L, 157S, 199V, 202K, 202L, 202N, 202R, 202T, 222C, 222P, 225D, 227A, 229C, 266N, 266N / 372T / 536T / 615D / 763L / 777G, 266N / 372T / 924N, 266N / 536T / 615D / 795E, 344G, 344M, 348G, 390Q / 615D, 412Y, 423V, 425R / 678I / 894C, 430F, 446T, 484L, 488G, 488K, 488M, 496G, 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, 580I, 580L, 580W, 580Y, 581F, 581G, 581H, 581L, 581S, 581T, 581V, 581Y,at least one substitution at a position or set of positions selected from 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, 691F, 691V, 692C, 692F, 692G, 692I, 692L, 692R, 692S, 692V, 692Y, 693F, 693I, 693Y, 705M, 706F, 706M, 706V, 708C, 709S, 710K, 710L, 710M, 710N, 710S, 711C, 711F, 711G, 711H, 711L, 711R, 711W, 726E, 768S, 773V, 777G, 777I, 777R, 779E, 779H, 779R, 795E, 797L, 797M, 816L, 826G, 834H, 857T, 859T, 859Y, 868I, 868L, 869L, 869S, 871E, 871K, 871R, 873A, 873F, 873Y, 877L, 877V, 878A, 878F, 878G, 878K, 878L, 878Q, 878R, 878S, 878W, and 909F, these positions being numbered with reference to SEQ ID NO: 1956. In some embodiments, the acid alpha-glucosidase is 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, D87E / 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, D348G, L390Q / Q615D, G412Y, L423V, Q425R / S678I / G894C, Y430F, S446T, N484L, L488G, L488K, L488M, A496G, H499Y / A711F, P503S, P503T, L530V, T543C, T543Q, T543S, T543V, L569H, L569I, L569Q, L569S, L569T, L569V, L569Y, A572G, A572S, I573C, I573D, I573H, I573M, I573Q, A574S, R577A, R577D, R577E, R577M, R577T, R577V, A578S, L579V, V580E, V580G, V580I, V580L, V580W, V580Y, K581F, K581G, K581H, K581L, K581S, K581T, K581V, K581Y, R583C, R583G, R583K, R583L, T585F, T585L, T585M, T585Q, T585V, F588L, F588V, V589I / M663F, Q615G, L628I, L628M, L628V, L629A, L629C, L629G, L629I, V631I, V631L, V631M, L633V, L656M, L656V, M663A, M663F, L669R, L670E, L670F, L670Icomprises at least one substitution at a position or set of positions selected from L670Q, L670R, L670S, L670T, L670V, L670W, S671A, S671G, S671M, S671T, S678H, S678L, S678T, S678Y, F679W, M687L, A690V, L691F, L691V, T692C, T692F, T692G, T692I, T692L, T692R, T692S, T692V, T692Y, L693F, L693I, L693Y, F705M, H706F, H706M, H706V, A708C, H709S, V710K, V710L, V710M, V710N, V710S, A711C, A711F, A711G, A711H, A711L, A711R, A711W, D726E, Q768S, E773V, S777G, S777I, S777R, P779E, P779H, P779R, Q795E, V797L, V797M, I816L, T826G, A834H, L857T, V859T, V859Y, V868I, V868L, I869L, I869S, L871E, L871K, L871R, R873A, R873F, R873Y, I877L, I877V, V878A, V878F, V878G, V878K, V878L, V878Q, V878R, V878S, V878W and L909F, these positions being numbered with reference to SEQ ID NO: 1956. In some embodiments, the acid alpha-glucosidase comprises at least one substitution at a position selected from 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, 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, these positions being numbered with reference to SEQ 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, At least one substitution is included at positions selected from 2W, 156C, 156K, 156R, 156S, 162T, 176R, 177Q, 185L, 186H, 187I, 199A, 199G, 199I, 199R, 199T, 199V / 775I, 199W, 202A, 202D, 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, 493L, 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, 765W, 768I, 768K, 768V, 773P, 777M, 777W, 779I, 779M, 779S, 788A, 788H, 788I, 788L, 788N, 788Q, 788S, 788T, 788Y, 797E, 797F, 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, and these positions are numbered with reference to SEQ ID NO: 1956. In some embodiments, the acid alpha-glucosidase is 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,Including at least one substitution at positions selected from T141R, A142G, T145A, T145L, F152L, F152S, F152W, I156C, I156K, I156R, I156S, D162T, K176R, D177Q, V185L, P186H, L187I, P199A, P199G, P199I, P199R, P199T, P199V / L775I, P199W, S202A, S202D, S202G, S202H, S202Q, S202Y, 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, N484A, N484K, N484R, L488C, L488E, L488S, D493L, A496M, A496W, H499A, H499E, 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, K752F, K752G, K752L, K752N, K752S, K752W, Y765W, Q768I, Q768K, Q768V, E773P, S777M, S777W, P779I, P779M, P779S, P788A, P788H, P788I, P788L, P788N, 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, and these positions areIt is numbered with reference to SEQ ID NO: 1956. In some embodiments, the acid alpha-glucosidase comprises at least one substitution at a position or set of positions selected from 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, and these positions are numbered with reference to SEQ ID NO: 1956. In some embodiments, the acid 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,comprises at least one substitution at a position or set of positions selected from 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, 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, these positions being numbered with reference to SEQ ID NO: 1956. In some embodiments, the acid 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, S136V, 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,Comprising at least one substitution at a position or set of positions selected from A572S, I573C, I573D, I573H, I573M, I573Q, A574S, R577A, R577D, R577E, R577T, L579V, V580E, V580G, V580W, K581G, K581H, K581T, R583C, R583G, T585Q, F588L, F588V, V589I / M663F, L628V, L629A, L629C, L629G, L629I, V631I, V631L, V631M, M663A, M663F, L669R, L670E, L670F, L670I, L670Q, L670R, L670S, L670T, L670V, L670W, 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, V797F, V797I, V797L, V797M, V797W, T826G, A834G, A834H, A834S, A834W, E855G, S856G, L857A, L857E, L857S, L857T, L857V, I869L, I869S, L871E, L871K, R873A, R873F, R873Y, V878A, V878G, V878K, V878Q, V878S, V878W, L909F, T924A, V926M, and V926T, and these positions are numbered with reference to SEQ ID NO: 1956.,

[0017] The present invention provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to the amino acid sequence numbered with reference to SEQ ID NO: 2496. In some embodiments, the acidic 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, 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 / 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,at least one substitution in a position or set of positions selected from 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 / 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, and these positions are numbered with reference to SEQ ID NO: 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, 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 / 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 / 711H, 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 / 869L / 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 / 869Scontains at least one substitution at a position or set 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, and these positions are numbered with reference to SEQ ID NO: 2496. In some embodiments, the acid 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 / A711H / 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, L569T / F588L, L569T / F588L / V589I / L628M / L629I / T692Y / A711H, L569T / F588L / S678T / T692Y, L569T / F588L / A711H / I869L / L871K / V878S, L569T / F588L / V878S, L569T / V589I / L628M / L670F / T692G / A711H, L569T / V589I / S678T / L871K / V878S, L569T / V589I / T692G / Q795E / L871K / V878S, L569T / V589I / L871S, L569T / 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 / 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 / A711HT692G / 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, contains at least one substitution at 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, these positions being numbered with reference to SEQ ID NO: 2496. In some embodiments, the acid alpha-glucosidase contains at least one substitution at a position or set of positions selected from 60 / 589, 307, 313, 584, and 810, these positions being numbered with reference to SEQ ID NO: 2496. In some embodiments, the acid alpha-glucosidase contains at least one substitution at a position or set of positions selected from 60V / 589A, 307T, 313T, 584C, and 810V, these positions being numbered with reference to SEQ ID NO: 2496. In some embodiments, the acid alpha-glucosidase contains at least one substitution at a position or set of positions selected from A60V / V589A, S307T, V313T, G584C, and L810V, these positions being numbered with reference to SEQ ID NO: 2496. In some embodiments, the 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,At least one substitution is included at a position or set of positions selected from 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 / 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 / 878, 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, 810, 812, 871, and 878, and these positions are numbered with reference to SEQ ID NO: 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 / 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 / 711H, 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, 584C, 584E, 585K, 588L / 589I / 628M / 678T, 589I, 589I / 670T / 692G / 795E / 871Kat least one substitution at a position or set of positions selected from 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 / 869L / 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 / 869S, 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, 810V, 812E, 871K, 871S, and 878S, where these positions are numbered with reference to SEQ ID NO: 2496. In some embodiments, the acid 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 / T692Y / A711H / 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, L569T / F588L,L569T / F588L / V589I / L628M / L629I / T692Y / A711H, L569T / F588L / S678T / T692Y, L569T / F588L / A711H / I869L / L871K / V878S, L569T / F588L / V878S, L569T / V589I / L628M / L670F / T692G / A711H, L569T / V589I / S678T / L871K / V878S, L569T / V589I / T692G / Q795E / L871K / V878S, L569T / V589I / L871S, L569T / 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 / 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, G584C, G584E, T585K, F588L / V589I / L628M / S678T, V589IV589I / 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 / L871K, T692Y, A711H, A711H / Q795E / I869S / V878S, A711H / I869S / V878S, A711H / L871K, Q795E / V878S, L810V, A812E, L871K, L871S, and at least one substitution at a position or set of positions selected from V878S, wherein these positions are numbered with reference to SEQ ID NO: 2496.

[0018] The present invention provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO: 2880, wherein the positions are numbered with reference to SEQ ID NO: 2880. In some embodiments, the acidic 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 / 894 / 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 / 50 / 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 / 883 / 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、at least one substitution at a position or set of positions selected from 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, 527 / 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, wherein these positions are numbered with reference to SEQ ID NO: 2880. In some embodiments, the acid alpha-glucosidase comprises at least one substitution at a position or set of positions selected from __, wherein these positions are numbered with reference to SEQ ID NO: 2880. In some embodiments, the 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 / 894Q / 913V, 24L / 28L / 50Q / 135S / 150T / 437A / 522E / 527N / 871L / 883R / 894Q / 932S, 24L / 28L / 62A / 522E / 569L / 932S, 24L / 28L / 437A / 486T / 527N, 24L / 29L / 39P / 50Q / 62A / 78P / 87D / 135S / 150T / 267R / 437A / 486T / 522E / 527N / 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 / 894Q, 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 / 551V / 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 / 527N / 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 / 727S / 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 / 527N / 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, 39P / 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 / 736M527N / 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 comprises at least one substitution at 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 SEQ ID NO: 2880. In some embodiments, the acid 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 / K267R / V522E / R527N / A551V / T569L / W727S / K830Q / S842G / E871L / H883R / G894Q / R913V, W24L / S28L / V50Q / Q135S / S150T / G437A / V522E / 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 / H883R / G894Q / R913V / A932S, W24L / V50Q / E78P / E87D / Q135S / S150T / K267R / E486T / V522E / R527N / A551V / T670L / W727S / P750A / K830Q / S842G / E871L / H883R / G894Q / R913V / A932S, W24L / V50Q / E486T / R527N / H711A / W727S, W24L / L62A / E87D / E486T / W727S, W24L / L62A / W727S / 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 / W727S / P750A / S842G / E871L, S28L, S28L / V50Q / E78P / E87D / Q135S / N266T / K267R / G437A / E486T / R527N / A551V / T670L / W727S / P750A / K830Q / S842G / E871L / H883R / G894Q / R913V / A932S, S28L / V50Q / V522E / R527N / H711A / W727S / E871L, 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, T29L / L62A / G437A / R527N, T29L / E78P / E87D / S150T / R527N / W727S, T29L / E78P / Q135S / W727S / K830Q, T29L / E87D, T29L / Q135S / S150T / R527N / T670L / W727S / H883R, T29L / S150T / K267R / W727S / P750A / E871L / H883R / A932S, T29L / S150T / G437A / W727S, T29L / V522E / T670L / H711A / E871L, T29L / T670L / A932S, Q39P / V50Q, Q39P / W727S / P750A / A932S, V50Q / Q135S / S150T / A932S, V50Q / G437A / V522E / R527N, V50Q / H711A, V50Q / W727S / P750A / H883R / G894Q, L62A, L62A / E87D / S150T, L62A / E87D / S150T / W727S, L62A / Q135S / V522E / H711A / W727S / P750A / S842G / E871L / G894Q, L62A / G437A, L62A / G437A / W727SE78P / 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,comprises at least one substitution at a position or set of positions selected from 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, 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, wherein these positions are numbered with reference to SEQ ID NO: 2880. In some embodiments, the 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 / 812 / 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,and contains at least one substitution at a position or set of positions selected from 692 / 812, these positions being numbered with reference to SEQ 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 / 736M / 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 / 878S, 527N / 727S / 736M, 527N / 736M / 932S, 527N / 812E, 678T / 692G / 727S / 812E / 842G, 678T / 692G / 812E, 678T / 812E, 692G / 727S / 736M / 842G / 913V, and contains at least one substitution at a position or set of positions selected from 692 / 812, these positions being numbered with reference to SEQ ID NO: 2880. In some embodiments, the acid alpha-glucosidase is 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 / A932S, 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 / S842G, S678T / T692G / A812E, S678T / A812E, T692G / W727S / L736M / S842G / R913V, and at least one substitution at a position or set of positions selected from T692G / A812E, where these positions are numbered with reference to SEQ ID NO: 2880.

[0019] The present invention provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO: 3104, wherein the positions are numbered with reference to SEQ ID NO: 3104. In some embodiments, the acidic alpha-glucosidase comprises at least one substitution at a position or set of positions selected from 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, 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, and these positions are numbered with reference to SEQ ID NO: 3104. In some embodiments, the acidic alpha-glucosidase is 62A, 62A / 89D / 830Q,At least one substitution is included at a position or set of positions selected from 62A / 248H / 678T / 830Q / 878S / 932S, 62A / 678T, 62A / 678T / 785Q, 62A / 678T / 830Q, 62A / 678T / 830Q / 860F, 62A / 678T / 830Q / 860F / 871L / 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, 62A / 830Q, 62A / 830Q / 860F, 62A / 830Q / 860F / 871L / 873H, 62A / 830Q / 860F / 878S / 932S, 62A / 830Q / 860F / 932S, 62A / 830Q / 871L / 932S, 62A / 830Q / 878S, 62A / 830Q / 932S, 62A / 833I / 860F / 932S, 62A / 860F, 62A / 860F / 871L, 62A / 860F / 871L / 878S, 62A / 860F / 871L / 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, and these positions are numbered with reference to SEQ ID NO: 3104. In some embodiments, the acid 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,At least one substitution at a position or set of positions selected from L62A / S678T / K830Q / L860F / E871L / V878S / A932S, L62A / S678T / K830Q / L860F / V878S, L62A / S678T / K830Q / L860F / V878S / A932S, L62A / S678T / 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 / A932S, L62A / S678T / V878S / A932S, L62A / S678T / V878S / A932T, L62A / S678T / A932S, L62A / K830Q, L62A / K830Q / L860F, L62A / K830Q / L860F / E871L / R873H, L62A / K830Q / L860F / V878S / A932S, L62A / K830Q / L860F / A932S, L62A / K830Q / E871L / A932S, L62A / K830Q / V878S, L62A / K830Q / A932S, L62A / M833I / L860F / A932S, L62A / L860F, L62A / L860F / E871L, L62A / L860F / E871L / V878S, L62A / L860F / E871L / A932S, L62A / L860F / 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, and these positions are numbered with reference to SEQ ID NO: 3104.,

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

[0021] In some embodiments, the recombinant acidic alpha-glucosidase comprises 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, the recombinant acidic alpha-glucosidase is derived from human acidic alpha-glucosidase. In some further additional embodiments, the recombinant acidic alpha-glucosidase comprises the polypeptide sequences 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 thermostable. In some further embodiments, the recombinant acid alpha-glucosidase exhibits resistance to proteolysis. In still some additional 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 at acidic pH and neutral pH. In still some additional embodiments, the recombinant acid alpha-glucosidase is purified. In some further embodiments, the recombinant acid alpha-glucosidase exhibits at least one improved property selected from 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) as compared to a reference sequence. In some further embodiments, the recombinant acid alpha-glucosidase exhibits at least one improved property selected from i) enhanced catalytic activity, ii) increased resistance to pH 7, iii) increased resistance to pH 4, iv) increased expression, v) increased uptake into cells, vi) increased enzymatic activity in cell lysates, vii) decreased immunogenicity, or any combination of i), ii), iii), iv), v), vi), and / or vii) as compared to a reference sequence. In some embodiments, the reference sequence is selected from SEQ ID NO: 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-glucosidase of SEQ ID NO: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104.In still some other embodiments, the recombinant acidic alpha-glucosidase is more stable than the acidic alpha-glucosidase of SEQ ID NO: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104 at pH 4. In some further embodiments, the recombinant acidic alpha-glucosidase shows an increase in expression as compared to the acidic alpha-glucosidase of SEQ ID NO: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104. In still some additional embodiments, the recombinant acidic alpha-glucosidase has higher lysosomal stability than the acidic alpha-glucosidase of SEQ ID NO: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104. In some further embodiments, the recombinant acidic alpha-glucosidase is more readily taken up by cells than the acidic alpha-glucosidase of SEQ ID NO: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104. In some additional embodiments, the recombinant acidic alpha-glucosidase exhibits greater enzymatic activity than the acidic alpha-glucosidase of SEQ ID NO: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104 in cell lysates. In some additional embodiments, the recombinant acidic alpha-glucosidase shows a decrease or reduction in immunogenicity as compared to the acidic alpha-glucosidase of SEQ ID NO: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104. In some embodiments, the recombinant acidic 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 to at least one of the even-numbered sequences of SEQ ID NOs: 8-3378. In some further embodiments, the recombinant acid alpha-glucosidase 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 to at least one of the even-numbered sequences of SEQ ID NOs: 8-3378. 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 of SEQ ID NOs: 8-3378. In some further embodiments, the recombinant acid alpha-glucosidase comprises a polypeptide sequence comprising at least one of the even-numbered sequences of SEQ ID NOs: 2-3378. In some further embodiments, the recombinant acid alpha-glucosidase consists of a polypeptide sequence comprising at least one of the even-numbered sequences of SEQ ID NOs: 8-3378.

[0024] The present invention also provides a composition 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 a recombinant polynucleotide sequence 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 to at least one of the even-numbered sequences of SEQ ID NOs: 8 to 3378. In some further embodiments, the recombinant acid alpha-glucosidase encoded by the 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 to at least one of the even-numbered sequences of SEQ ID NOs: 8 to 3378.

[0026] In some further embodiments, the recombinant polynucleotide sequence has 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 to at least one of the odd-numbered sequences of SEQ ID NOs: 7 to 3377. In some further embodiments, the recombinant polynucleotide sequence is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to at least one of the odd-numbered sequences of SEQ ID NOs: 7 to 3377. In some additional embodiments, the recombinant polynucleotide sequence comprises a sequence having at least 90% sequence identity to at least one of the odd-numbered sequences of SEQ ID NOs: 7 to 3377. In still some further embodiments, the recombinant polynucleotide sequence comprises an odd-numbered sequence of SEQ ID NOs: 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 linked 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 herein referred to 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 the acid alpha-glucosidase provided herein. In some further embodiments, the pDH vector comprises at least one polynucleotide sequence selected from the odd-numbered sequences of 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 the even-numbered sequences of SEQ ID NOs: 2 to 3378. In some embodiments, the pDH vector comprises SEQ ID NO: 3379, while in some other embodiments, the pDH vector comprises SEQ ID NO: 3380. In some additional embodiments, SEQ ID NO: 1 contained in SEQ ID NO: 3379 is replaced with another polynucleotide sequence. In some embodiments, SEQ ID NO: 1 in SEQ ID NO: 3379 is replaced with at least one polynucleotide sequence selected from the odd-numbered sequences of SEQ ID NOs: 1 to 3377. In some additional embodiments, the pDH vector comprises the plasmid provided in the map in FIG. 9, while in some other embodiments, the pDH vector comprises the plasmid provided in the map in FIG. 10. In some embodiments, the "stuffer sequence" (i.e., the bla sequence) in the plasmid shown in FIG. 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 at base pairs 724 to 1581 (for a total of 858 base pairs). As used herein, the terms "stuffer gene" and "stuffer sequence" refer to sequences within a plasmid vector that are replaced with the gene of interest.As used herein, the term "gene of interest" refers to a gene encoding a desired polypeptide (e.g., a "polypeptide of interest"). In some additional embodiments, the stuffer sequence is replaced with a gene of interest (i.e., a gene for which expression is desired for the production of a polypeptide of interest such as a variant acid alpha-glucosidase).

[0028] The present invention also provides a host cell comprising at least one expression vector provided herein. In some embodiments, the expression vector harbored 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 a recombinant acid alpha-glucosidase variant, the method comprising culturing at least one host cell provided herein under conditions in which an acid alpha-glucosidase encoded by a recombinant polynucleotide is produced. 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 a recombinant acid alpha-glucosidase variant produced according to the method provided herein.

[0030] The present invention also provides a composition comprising at least one recombinant acid alpha-glucosidase provided herein. The present invention also encompasses the use of the composition 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 a human. In some further additional embodiments, the present invention provides a pharmaceutical composition comprising at least one recombinant polynucleotide provided herein. In some 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, the method comprising the steps of providing 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 Description of the Drawings

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[0059] Description of the Invention The present invention provides engineered acidic 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 susceptibility to proteolysis. The present invention also provides methods for using compositions comprising the engineered GAA polypeptides for therapeutic and other purposes. Abbreviations and Definitions:

[0060] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. 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 those well known and commonly employed in the art. Such techniques are well known and described in numerous textbooks and references well known to those of skill in the art. Standard techniques or their modified forms are used in chemical synthesis and chemical analysis. All patents, patent applications, articles, and published literature referred to herein above and below are hereby expressly incorporated herein by reference.

[0061] Any suitable methods and materials similar or equivalent to those described herein are used in the practice of the present invention, but some methods and materials are described herein. It is to be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described. These methodologies, protocols, and reagents may vary depending on the circumstances in which they are used by those of ordinary skill in the art. Accordingly, the terms defined immediately below are more fully described by reference to the entire present application. All patents, patent applications, articles, and published literature referred to herein above and below are hereby expressly incorporated herein by reference.

[0062] Also, as used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0063] Numeric ranges are inclusive of the numbers defining the ranges. Thus, all numeric ranges disclosed herein are intended to include any and all narrower numeric ranges that fall within such broader numeric ranges as if such narrower numeric ranges were all expressly written herein. Any maximum (or minimum) numeric limitation disclosed herein is also intended to include any and all lower (or upper) numeric limits as if such lower (or upper) numeric limits were expressly written herein.

[0064] The term "about" means an acceptable error for a particular value. In some instances, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% of a given value range. In some instances, "about" means within 1, 2, 3, or 4 standard deviations of a given value. In some instances, "about" includes 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 given herein do not limit the various aspects or embodiments of the invention that can be understood by reference to the present application as a whole. Thus, the terms defined immediately below are more fully defined by reference to the present application as a whole. Nevertheless, for ease of understanding, some terms are defined below.

[0066] Unless otherwise indicated, each nucleic acid is described in the 5' to 3' direction, left to right, and each amino acid sequence is described in the amino to carboxy direction, left to right.

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

[0068] The "EC" numbers relate 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 ), and its biorepository collection includes genes and strains.

[0070] "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

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

[0072] As used herein, "Pompe disease" refers to type II glycogen storage disease, an autosomal recessive genetic disorder that results in a metabolic disorder characterized by lysosomal accumulation of glycogen in skeletal muscle and other tissues. Pompe disease is characterized based on age of onset, organ lesions, severity, and rate of progression. The more severe form is infantile-onset Pompe disease (IOPD), which occurs in infancy. Another form, called "late-onset Pompe disease" (LOPD), occurs in individuals who develop the disease before 12 months of age but without cardiomyopathy associated with IOPD, and in all individuals who develop the disease after 12 months of age. Synonyms for Pompe disease include "acid alpha-glucosidase deficiency", "acid maltase deficiency", "GAA deficiency", "type II glycogen storage disease", "GSD II", "GSD2", and "type II glycogenosis".

[0073] As used herein, the terms "protein", "polypeptide", and "peptide" are used interchangeably to denote a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation).

[0074] The term "amino acid" is referred to herein by either their generally known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Nucleotides can likewise be referred to by their generally accepted one-letter codes.

[0075] The terms "engineered", "recombinant", "non-naturally occurring", and "variant", when used with respect to a cell, polynucleotide or polypeptide, refer to a material or a material corresponding to the native or natural form of that material that has been modified in a manner that does not occur naturally or that is identical but produced or derived by an operation using synthetic materials and / or 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 exists in an organism, can be isolated from a natural source, and has not been intentionally modified by human manipulation.

[0077] A "coding sequence" refers to the portion of a nucleic acid (e.g., a gene) that encodes the amino acid sequence of a protein.

[0078] The term "percent sequence identity" is used herein to refer to the comparison between polynucleotides and polypeptides and is determined by comparing two optimally aligned sequences over a comparison window, where a portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence for the optimal alignment of these two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues occur 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 percent sequence identity. Alternatively, the percentage can be calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues occur in both sequences or the number of positions at which the nucleic acid bases or amino acid residues are aligned using gaps 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 percent sequence identity. It is understood by those skilled in the art that there are numerous established algorithms available for aligning two sequences. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math. 2:482

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

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

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

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

[1977] ). 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-valued threshold score T when aligned with words of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., see above (to be illuminated). These initial adjacent word hits serve as seeds to initiate a search to find longer HSPs that contain them. Thus, the word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. The cumulative score is calculated for nucleotide sequences using parameters M (reward score for a pair of matching residues; always, >0) and N (penalty score for a mismatched residue; always, <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the word hit in each direction stops when the cumulative alignment score drops by an amount X from its maximum achieved value; stops when the cumulative score becomes zero or less due to the accumulation of one or more negatively scored residue alignments; or stops when the end of either sequence is reached. The sensitivity and speed of the alignment are determined by the BLAST algorithm parameters W, T, and X. The BLASTN program (for nucleotide sequences) uses, by default, a word length (W) of 11, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program uses, by default, a word length (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

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

[0079] As used herein, the term "reference sequence" refers to a defined sequence used as a basis for sequence comparison. The reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. In general, a reference sequence is at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length, or full-length, of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) contain sequences that are similar between the two sequences (i.e., a portion of the full sequence) and (2) further contain sequences that differ between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is usually performed by comparing the sequences of the two polynucleotides or polypeptides over 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 changes from the primary sequence. A "comparison window" is a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues, such that a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and the portion of the sequence within the comparison window of that sequence contains 20 percent or less addition or deletion (i.e., gap) compared to the reference sequence (including no addition or deletion) for optimal alignment of the two sequences. The comparison window may be longer than 20 contiguous residues and may include windows of 30, 40, 50, 100, or longer as needed.

[0080] "Corresponding to," "reference to," or "compared to," when used in the context of numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of the designated reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is indicated relative to a reference sequence rather than by the actual numbered position 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 engineered GAA, can be aligned with a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, gaps are present, but the numbering of the residues in the given amino acid or polynucleotide sequence is done relative to the reference sequence to which it is aligned.

[0081] "Amino acid difference" or "residue difference" refers to the difference of an amino acid residue at a position in a polypeptide sequence compared to the amino acid residue at the corresponding position in a reference sequence. The position of the amino acid difference is generally referred to herein as "Xn", where n in this case refers to the corresponding position in the reference sequence that serves as the basis for the residue difference. For example, "a residue difference at position X27 compared to SEQ ID NO:2" refers to the difference of the amino acid residue at the polypeptide position corresponding to position 27 of SEQ ID NO:2. Thus, if the reference polypeptide of SEQ ID NO:2 has phenylalanine at position 2, "a residue difference at position X27 compared to SEQ ID NO:2" is an amino acid substitution of any residue other than phenylalanine at the position of the polypeptide corresponding to position 27 of SEQ ID NO:2__. In most examples herein, a specific amino acid residue difference at a position is indicated as "XnY", where "Xn" designates the corresponding position as described above, and "Y" is the one-letter identifier of the amino acid found in the engineered polypeptide (i.e., a residue different from that in the reference polypeptide). In some examples (e.g., as shown 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 present disclosure also provides specific amino acid differences indicated by the conventional notation "AnB", where A is the one-letter identifier of the residue in the reference sequence, "n" is the number of the residue position in the reference sequence, and B is the one-letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some examples, the polypeptides of the present disclosure may include one or more amino acid residue differences compared to the reference sequence, and these differences are indicated by a list of the designated positions where residue differences exist compared to the reference sequence. In some embodiments where more than one amino acid can be used at the designated residue position of the polypeptide, the various amino acid residues that can be used are separated by " / " (e.g., X27P / X27R or X27P / R). In some embodiments, the enzyme variant includes more than one substitution.These substitutions are separated by slashes (e.g., F27P / C944W) for ease of reading. In some cases, "X" is not appended before the position number in the present application. The present application includes engineered polypeptide sequences that contain one or more amino acid differences, including either and / or both conservative amino acid substitutions and non-conservative amino acid substitutions.

[0082] "Conservative amino acid substitutions" refer to the substitution of a residue with a different residue having a similar side chain, and thus typically include the substitution of an amino acid in a polypeptide with an amino acid within the same or a similar defined class of amino acids. By way of example and not limitation, an amino acid having an aliphatic side chain can be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine), an amino acid having a hydroxyl side chain can be substituted with another amino acid having a hydroxyl side chain (e.g., serine and threonine), an amino acid having an aromatic side chain can be substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine), an amino acid having a basic side chain can be substituted with another amino acid having a basic side chain (e.g., lysine and arginine), an amino acid having an acidic side chain can be substituted with another amino acid having an acidic side chain (e.g., aspartic acid or glutamic acid), and / or a hydrophobic or hydrophilic amino acid can be replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0083] "Non-conservative substitutions" refer to the substitution of an amino acid in a polypeptide with an amino acid having significantly different side chain characteristics. Non-conservative substitutions can involve the use of amino acids from outside rather than within a defined group, and affect (a) the structure of the peptide backbone in the region of the substitution (e.g., substitution of glycine with proline), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions can be an acidic amino acid substituted with a basic or aliphatic amino acid, an aromatic amino acid substituted with a small amino acid, and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0084] "Deletion" refers to the modification of a polypeptide by the removal of one or more amino acids from a reference polypeptide. Deletions may involve 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 a maximum of 10% of the total number of amino acids, or up to a maximum of 20% of the total number of amino acids, from the reference enzyme while retaining the enzyme activity of the engineered enzyme and / or retaining improved properties. Deletions may relate to internal and / or terminal portions of the polypeptide. In various embodiments, deletions may include contiguous segments or may be discontinuous.

[0085] "Insertion" refers to the modification of a polypeptide by the addition of one or more amino acids to a reference polypeptide. Insertions may be within the internal portion of the polypeptide or may be at the carboxy or amino terminus. As used herein, insertions include fusion proteins as are known in the art. Insertions may be contiguous segments of amino acids in a naturally occurring polypeptide or may be separated by one or more amino acids.

[0086] As used interchangeably herein, "functional fragment" or "bioactive fragment" refers to a polypeptide having amino-terminal and / or carboxy-terminal deletions and / or internal deletions, wherein the remaining amino acid sequence is in the same position as the corresponding position in the sequence to which it is being compared (e.g., the full-length engineered 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 is substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that are naturally associated with it. This term encompasses polypeptides that have been removed or purified from their natural environment or expression system (e.g., host cell or in vitro synthesis). Recombinant GAA polypeptides may be present intracellularly, in the cell culture medium, or may be prepared in various forms such as lysates or isolated preparations. Therefore, in some embodiments, the recombinant GAA polypeptide can be an isolated polypeptide.

[0088] "Substantially pure polypeptide" refers to a composition in which the polypeptide species is the dominant species present (i.e., on a molar or weight basis, its abundance in the composition is greater than that of any other individual macromolecular species), and generally, when the species of interest constitutes at least about 50 percent of the macromolecular species present, on a molar or weight percentage basis, the composition is substantially purified. Generally, a substantially pure GAA composition constitutes about 60% or higher%, about 70% or higher%, about 80% or higher%, about 90% or higher%, about 95% or higher%, and about 98% or higher% of the total macromolecular species present in the composition. In some embodiments, the species of interest is purified to essential homogeneity where the composition consists essentially of a single macromolecular species (i.e., contaminating species cannot be detected in the composition by conventional detection methods). Solvent species, small molecules (<500 daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant GAA polypeptide is a substantially pure polypeptide composition.

[0089] "Improved enzymatic properties" refers to an engineered GAA polypeptide that exhibits an improvement in any enzymatic property, as 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, increased protein expression, increased thermal activity, increased thermal stability, increased pH activity, increased stability, increased enzymatic 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 resistance to acidic, neutral or basic pH, increased resistance to proteolytic activity (i.e., decreased susceptibility to proteolysis), reduced aggregation, increased solubility, decreased immunogenicity, improved post-translational modification (e.g., glycosylation), altered temperature profile, increased lysosomal stability, and the like.

[0090] "Increased enzymatic activity" or "enhanced enzymatic activity" refers to an improved property of an engineered GAA polypeptide that can be represented 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 starting amount of substrate converted to product within a specified period using a specified amount of GAA), as compared to a reference GAA enzyme. Exemplary methods for determining enzymatic activity are provided in the Examples. Any property related to enzymatic activity can be affected, and such properties include the classical enzymatic properties of K m , V max or k cat . The improvement in enzymatic activity can range from about 1.1-fold the enzymatic activity of the corresponding wild-type enzyme to an enzymatic activity that is 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or higher than that of the naturally occurring GAA or another engineered GAA from which the GAA polypeptide was derived.

[0091] In some embodiments, the engineered GAA polypeptide has a k of at least 0.1 / sec, at least 0.5 / sec, at least 1.0 / sec, at least 5.0 / sec, at least 10.0 / sec, and in some preferred embodiments greater than 10.0 / sec. cat In some embodiments, K m is in the range of about 1 μM to about 5 mM, in the range of about 5 μM to about 10 mM, in the range of about 30 μM to about 30 mM, or in the range of about 50 μM to about 50 mM. In some specific embodiments, the engineered GAA enzyme exhibits an improvement in enzyme activity that is within the range of 1.5- to 10-fold, 1.5- to 25-fold, 1.5- to 50-fold, 1.5- to 100-fold, or greater than 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 4-methylumbelliferyl-α-D-glucopyranoside (4-MUGlu) molecules. Comparison of enzyme activities 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 amount of protein assayed are determined, and the use of the same expression system and the same host cell is also determined to minimize variations in the amount of enzyme present in the lysates produced by the host cells.

[0093] The term "improved resistance to acidic pH" means that the recombinant GAA according to the present invention will have improved stability (more highly retained activity at about 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 resistance to neutral pH" means that the recombinant GAA according to the present invention will have improved stability (more highly retained activity at about 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 an increase in 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 (retaining more highly intracellular activity after incubation of the cultured cells over a specified period compared to a reference GAA or another enzyme). In some additional embodiments, the recombinant GAA provided herein exhibits intracellular activity that is more highly 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 about 4 hours, but in some other embodiments, the period is less than 4 hours (e.g., 1, 2, or 3 hours), and in some alternative embodiments, the period is longer than 4 hours (e.g., 5, 6, 7, 8, or longer hours).

[0096] The terms "decreased immunogenicity" and "reduced immunogenicity" mean that the recombinant GAA provided herein induces a decrease in the immune response compared to wild-type or another reference GAA.

[0097] "Physiological pH", as used herein, means the pH range generally found in the blood of a subject (e.g., human).

[0098] The term "basic pH" (e.g., used with respect to improvement in stability under basic pH conditions or increase in resistance to basic pH) means a pH range of about 7 to 11.

[0099] The term "acidic pH" (e.g., used with respect to improvement in stability under acidic pH conditions or increase in resistance to acidic pH) means a pH range of about 1.5 to 4.5.

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

[0101] "Hybridization stringency" relates to hybridization conditions, e.g., washing conditions, in the hybridization of nucleic acids. Generally, the hybridization reaction is carried out under conditions of lower stringency, followed by washing under various, but higher, stringency conditions. The term "moderately stringent hybridization" refers to conditions that allow a target DNA to bind to a complementary nucleic acid having about 60% identity to the target DNA, preferably about 75% identity, about 85% identity, and higher than about 90% identity to the target polynucleotide. Exemplary moderately stringent conditions correspond to hybridization in 50% formamide, 5× Denhardt's solution, 5× SSPE, 0.2% SDS at 42 °C, followed by washing in 0.2× SSPE, 0.2% SDS at 42 °C. "High stringency hybridization" is the thermal melting temperature T determined under solution conditions for a defined polynucleotide sequence. mGenerally refers to conditions that are about 10 °C or less. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only nucleic acid sequences that form stable hybrids at 65 °C in 0.018 M NaCl (i.e., if the hybrid is not stable at 65 °C in 0.018 M NaCl, it will not be stable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridization at 42 °C in conditions corresponding to 50% formamide, 5× Denhardt's solution, 5× SSPE, 0.2% SDS, followed by washing at 65 °C in 0.1× SSPE and 0.1% SDS. Another high stringency condition is hybridization in conditions corresponding 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 high stringency hybridization conditions, and moderately stringent conditions, are described in the references above.

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

[0103] The "control sequences" are herein referred to as encompassing all components necessary or advantageous for the expression of the polynucleotides and / or polypeptides of the present application. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leader, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, start sequences and transcription terminators. At a minimum, the control sequences include a promoter, as well as transcription and translation stop signals. The control sequences may also be provided with linkers to introduce specific restriction sites facilitating ligation of the control sequences to the coding region of the nucleic acid sequence encoding the polypeptide.

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

[0105] The "promoter sequence" refers to a nucleic acid sequence recognized by a host cell for the expression of a polynucleotide of interest, e.g., a coding sequence. The promoter sequence contains transcriptional control sequences that mediate the expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence showing transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and the promoter can be obtained from a gene encoding an extracellular or intracellular polypeptide, either homologous or heterologous to the host cell.

[0106] "Suitable reaction conditions" refers to the conditions (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffer, co-solvent, etc.) in the enzymatic conversion reaction solution under which the GAA polypeptide of the present application can convert a substrate into a desired product compound. Exemplary "suitable reaction conditions" are provided in the present application and illustrated by examples. "Loading" such as in "compound loading" or "enzyme loading" refers to the concentration or amount of a constituent in the reaction mixture at the start of the reaction. "Substrate" in the context of the enzymatic conversion reaction process refers to a compound or molecule that is acted upon by the GAA polypeptide. "Product" in the context of the enzymatic conversion process refers to a 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 suitable 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 wild-type polypeptide of interest can be cloned into a vector such as a plasmid and expressed in a desired host such as E. coli, S. cerevisiae, etc. Variants of the recombinant polypeptide can be generated by various methods known in the art. In fact, there are a wide variety of different mutagenesis techniques well-known to those skilled in the art. In addition, mutagenesis kits are also available from many commercial molecular biology suppliers. Methods for performing specific substitutions (site-directed) at defined amino acids, specific or random mutagenesis (site-specific) in local regions of the gene, or random mutagenesis throughout the gene (e.g., saturation mutagenesis) can be utilized. A very large number of suitable methods for generating enzyme variants are known to those skilled in the art, and such methods include site-directed mutagenesis of single-stranded or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation mutagenesis, or any other suitable method known in the art, but are not limited thereto. Non-limiting examples of methods used in DNA and protein engineering are provided in the following patents: U.S. Patent No. 6,117,679, U.S. Patent No. 6,420,175, U.S. Patent No. 6,376,246, U.S. Patent No. 6,586,182, U.S. Patent No. 7,747,391, U.S. Patent No. 7,747,393, U.S. Patent No. 7,783,428, and U.S. Patent No. 8,383,346. After producing the variants, they can be screened for any desired property (e.g., high activity or increased activity, or low activity or decreased activity, increased thermal activity, increased thermal stability, and / or acidic pH stability, etc.). In some embodiments, "recombinant GAA polypeptide" (also referred to herein as "engineered GAA polypeptide", "variant GAA enzyme", and "GAA variant") is 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 linked to suitable control sequences for effecting the expression of a polypeptide encoded by the DNA sequence in a suitable host. In some embodiments, an "expression vector" has a promoter sequence operably linked to a DNA sequence (e.g., a transgene) for driving expression in a host cell and, in some embodiments, also includes a transcription terminator sequence. In some preferred embodiments, the pDH vector provided herein is used.

[0110] As used herein, the term "gene therapy vector" refers to a vehicle or carrier suitable for delivery of a polynucleotide sequence into a cell. In some embodiments, the vector encapsulates a gene (e.g., a therapeutic gene) or polynucleotide sequence for delivery into a cell or tissue, and 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 is used, it is not intended that the present invention 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 can find more general utility.

[0111] As used herein, the term "expression" includes any step 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 a polypeptide from a cell.

[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 step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, this term also encompasses the secretion of a polypeptide from a cell.

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

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

[0115] The term "analog" means a polypeptide having a sequence identity higher than 70% but less than 100% with a reference polypeptide (e.g., sequence identity higher than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%). In some embodiments, the term analog refers to a polypeptide that contains 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 that is administered to a subject exhibiting signs or symptoms of a pathological condition and that has a beneficial or desirable medical effect.

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

[0118] The term "gene therapy" refers to the delivery of a gene, polynucleotide, or polynucleotide sequence by a gene therapy vector to a cell or tissue for the treatment or prevention of a disease or for the modification of those cells or tissues. Gene therapy can include replacing a mutant gene that causes a disease with a healthy copy of that gene, or inactivating or "knocking out" a mutant gene that is malfunctioning. In some embodiments, gene therapy is used for the treatment of a disease in a patient.

[0119] The term "mRNA therapy" refers to the delivery of an mRNA polynucleotide sequence to a cell or tissue for the treatment or prevention of a disease or for the modification of those cells or tissues. In some embodiments, the mRNA polynucleotide sequence for delivery to the cell or tissue is formulated, for example, but not limited to, in liposomes. In some embodiments, mRNA therapy is used for the treatment of a disease in a patient.

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

[0121] The term "effective amount" means an amount sufficient to produce a desired result. One of ordinary skill in the art can determine what an effective amount is by using routine experimentation.

[0122] The terms "isolated" and "purified" are used to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that has been removed from at least one other component with which it is naturally associated. The term "purified" does not require absolute purity, but rather is intended as a relative definition.

[0123] The term "subject" includes 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 females and males.

[0124] As used herein, the term "patient" means any subject who is to be evaluated for a disease, who is to be treated for a disease, or who is experiencing a disease.

[0125] The term "infant" refers to a child from about 1 month to about 1 year of age after birth. As used herein, the term "newborn" refers to a child from birth to 28 days after birth. The term "premature infant" refers to an infant who is more than 20 weeks of gestation but before full term, generally weighing about 500 - about 2499 grams at birth. A "very low birth weight infant" is an infant weighing less than 1500 g at birth.

[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, this 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 majority in the relevant jurisdiction (e.g., 18 years in the United States). In some embodiments, this term refers to any sufficiently grown and mature organism. In some embodiments, the term "young adult" refers to a person who is under 18 years old but has reached sexual maturity.

[0128] As used herein, "composition" and "formulation" encompass products (e.g., pharmaceutical compositions, health / nutritional supplements, feeds, etc.) containing at least one engineered GAA of the invention for any suitable use.

[0129] The terms "administer" and "administering" a composition mean providing the composition of the invention to a subject (e.g., a person affected by Pompe disease).

[0130] The term "carrier", when used in connection with a pharmaceutical composition, means any of a standard pharmaceutical carrier, buffer, and excipient, e.g., a stabilizer, preservative, and adjuvant.

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

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

[0133] The term "therapeutically effective amount", when used in connection with the symptoms of a disease / condition, refers to the amount and / or concentration of a compound (e.g., an engineered GAA polypeptide) that improves, attenuates, or eliminates one or more symptoms of the disease / condition, or prevents or delays the onset of the symptoms.

[0134] The term "therapeutically effective amount", when used in connection with a disease / condition, refers to the amount and / or concentration of a composition (e.g., an engineered GAA polypeptide) that ameliorates, attenuates or eliminates the disease / condition. In some embodiments, the term is used in connection with the amount of a composition that elicits a biological (e.g., medical) response in a tissue, system or animal subject, as sought by a researcher, physician, veterinarian or other clinician.

[0135] The terms "treating", "treat" and "treatment" are intended to include not only palliative treatment but also prophylactic (e.g., preventive) treatment. Engineered GAA polypeptide:

[0136] In some embodiments, an engineered GAA polypeptide is produced by culturing a microorganism comprising 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 tables showing sequence structure information demonstrating the correlation between specific amino acid sequence features and the functional activity of the engineered GAA polypeptides. 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 activity data determined by experiments on exemplary engineered GAA polypeptides. Polynucleotides, expression vectors and host cells encoding engineered polypeptides:

[0138] The present invention provides a polynucleotide encoding an engineered GAA polypeptide described herein. In some embodiments, the polynucleotide is operably linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. An expression construct containing a heterologous polynucleotide encoding an engineered GAA polypeptide can be introduced into a suitable host cell to express the corresponding GAA polypeptide.

[0139] As will be apparent to those skilled in the art, knowledge of the protein sequence and of the codons corresponding to the various amino acids allows one to describe all the polynucleotides capable of encoding the polypeptide of interest. The degeneracy of the genetic code, whereby the same amino acid is encoded by alternative or synonymous codons, allows one to generate a very large number of nucleic acids, all of which encode an engineered GAA polypeptide. Thus, given knowledge of a particular amino acid sequence, one skilled in the art can generate any number of different nucleic acids by simply modifying the sequence of one or more codons in a way that does not change the amino acid sequence of the protein. In this context, the present invention specifically contemplates each and every possible variant of the polynucleotides encoding the polypeptides described herein, such variants being made by selecting combinations based on possible codon choices, and all such variants are considered to be specifically disclosed herein for any of the 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, and SEQ ID NOs: 2, 8 and / or 14.

[0140] In various embodiments, the codons are preferably selected to be compatible with the host cell in which the protein is to be produced. For example, the preferred codons used in bacteria are those used for expression in bacteria. Thus, a codon-optimized polynucleotide encoding an engineered GAA polypeptide contains 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 with the properties disclosed herein, having an amino acid sequence that has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to a reference sequence selected from SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104, or an 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, and contains one or more residue differences as 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 an 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., at 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 SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104.

[0142] The present invention provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO:2. The present invention provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO:2. In some embodiments, the acidic alpha-glucosidase comprises at least one substitution at a position or set of positions selected from 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, 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, these positions being numbered with reference to SEQ ID NO:2. In some embodiments, the acidic alpha-glucosidase is 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,including 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, and these positions are numbered with reference to SEQ ID NO: 2. In some embodiments, the acid alpha-glucosidase includes at least one substitution or set of substitutions at one or more positions selected from F27P, F27P / C944W, F27R, L28P, L28R, L28S, L29T / A478T, V30G, V30K, V30T, K88G, K88S, Q107G, Q107P, L109G / G842E, L109P, Q110G, Q110L, Q113S, S135A, S135Q, E137P, M138A, T148G, T148Y, T150G, Q247R, D274G, A276F, A276Y, T278A, T278G, I375E, R403W, R414P, A418E / H499R, A418R, Q421S, G426R, A437S, A444T, R455V, 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, and these positions are numbered with reference to SEQ ID NO: 2. In some embodiments, the 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、including at least one substitution at a 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, these The position is numbered with reference to Array No. 2. In some embodiments, the 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 / 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 / 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 / 610RComprising at least one substitution or set of substitutions at one or more positions 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, these positions being numbered with reference to SEQ 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 / 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 / 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 / S727PK106P / 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 / C944SH191R / 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 / P922LL275V / 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 / S671Nand comprising at least one substitution or set of substitutions at one or more positions selected from A547G / V638I / S668D, these positions being numbered with reference to SEQ ID NO: 2.,

[0143] The present invention provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO:8. The present invention provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO:8. In some embodiments, the acidic 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,at least one substitution at 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, these positions being numbered with reference to SEQ ID NO: 8. 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 / 749E, 106P / 218S / 281V, 106P / 218S / 455V, 106P / 218S / 455V / 507L / 749E, 106P / 489R / 671N, 106P / 638I, 106P / 671N / 934R, 113S, 113S / 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 / 281V, 218S / 281V / 431V, 218S / 281V / 671N, 218S / 431V, 218S / 431V / 489R / 507L / 749E / 934R, 218S / 455V, 218S / 507L / 749E, 218S / 507L / 934R, 218S / 638I / 671N, 218S / 749E, 281V / 431V / 489R / 668D, 345K / 934R, 418E, 418E / 489R, 431V / 668D / 671N,Comprising 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, these positions being numbered with reference to SEQ ID NO: 8. 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, 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,Comprising at least one substitution or set of substitutions at one or more positions selected from 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 Q749E / A784, these positions being numbered with reference to SEQ ID NO: 8.,

[0144] The present invention provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO: 14. The present invention provides a recombinant acidic alpha-glucosidase and / or a bioactive recombinant acidic alpha-glucosidase fragment 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 to SEQ ID NO: 14. In some embodiments, the acidic 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 / 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, 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 / 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,Comprising at least one substitution at a position or set of positions selected from 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, 448, 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, these positions being numbered with reference to SEQ ID NO: 14. In some embodiments, the 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 / 793K, 37F / 196T, 37F / 528S / 696S / 793K, 37F / 528S / 790V, 37F / 528S / 790V / 793K / 862Q, 37F / 790V / 793K, 39D, 39H, 39Q, 39Q / 58L / 489D / 725E / 830K / 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,comprising at least one substitution or set of substitutions at one or more positions selected from 944S, these positions being numbered with reference to SEQ ID NO: 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 / K154R / D523N / E681Q / E793K / R862Q, S37F / N79S / K154R / E793K, S37F / A196T, S37F / N528S / A696S / E793K, S37F / N528S / I790V, S37F / N528S / I790V / 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 / Q830K / 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, A62E / N79S / R862Q, A62E / Q92R, A62E / Q92R / I790V / E793K, A62E / K106R / D523N / N528S / A696S / E793K / R862Q, A62E / K154R / A696S / E793K / R862Q,One or more substitutions or sets of substitutions at one or more positions selected from A62E / E793K / R862Q, R68N, R68S, R68W, V70A / R267K / K725E / C944S, V70A / R267K / C930P / C944S, V70A / A489D / C930P, V70A / K725E / Q830K / L860F / C930P / C944S, V70Q, P77W, N79S / K154R / E681Q, N79S / K154R / E793K / R862Q, N79S / R862Q, A89R, A97D, A97G, K106R / 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, E208G, E208I, Q217A, Q217D, R267K / A489D / D500A / K725E / Q830K / C930P, R267K / E522V / K725E, Y352K, Y352V, R385G, H424K, R448L, E463A, A489D / Q830K / C944S, D500A, D500A / S612D / Q830K / L860F, D500A / L860F / C930P, D500A / C930P / C944S, E522V / K725E, D523N, D523N / I790V / E793K, N528S / E681Q, N528S / E793K, N528S / R862Q, L672E, L672K, P673N, P673R, K725F, 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, these positions being numbered with reference to SEQ ID NO: 14.,

[0145] In some embodiments, the recombinant acid alpha-glucosidase comprises 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, the recombinant acid alpha-glucosidase is derived from human acid alpha-glucosidase. In yet some additional embodiments, the recombinant acid alpha-glucosidase comprises the polypeptide sequences of SEQ ID NO: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104.

[0146] In some embodiments, the polynucleotide encoding the engineered 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 engineered 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 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 engineered GAA polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide residue identity 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 engineered GAA polypeptide comprises 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 engineered 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, the polynucleotide can hybridize 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 the complementary sequence thereof, or a polynucleotide sequence encoding any of the variant GAA polypeptides provided herein.

[0147] In some embodiments, an isolated polynucleotide encoding any of the engineered GAA polypeptides provided herein is engineered in various ways to effect polypeptide expression. In some embodiments, a polynucleotide encoding a polypeptide is provided as an expression vector in which one or more control sequences are present to regulate the expression of the polynucleotide and / or the polypeptide. Manipulation of the isolated polynucleotide prior to its insertion into the vector may or may not be desirable 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 control array includes, among other arrays, a promoter, Kozak sequence, leader sequence, polyadenylation sequence, propeptide sequence, signal peptide sequence, DNA-based regulatory element for gene therapy retention, and a transcription terminator. As is known in the art, suitable promoters can be selected based on the host cell used. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present application include the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltose-producing amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase genes (see, e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75: 3727-3731

[1978] ), as well as the tac promoter (see, e.g., DeBoer et al., Proc. Natl Acad. Sci. USA 80: 21-25

[1983] ), but are not limited thereto.Exemplary promoters for filamentous fungal host cells include promoters obtained from the genes of Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic 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, e.g., WO96 / 00787), as well as the NA2 - tpi promoter (a hybrid of the promoter from the gene of Aspergillus niger neutral alpha - amylase and the promoter from the gene of Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters are Saccharomyces cerevisiae enolase (ENO - 1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde - 3 - phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae. It may be derived from the gene of 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] ). Exemplary promoters for use in mammalian cells include those from cytomegalovirus (CMV), the chicken β-actin promoter fused to the CMV enhancer, simian vacuolating virus 40 (SV40), Homo sapiens phosphoglycerate kinase, beta-actin, elongation factor-1a or glyceraldehyde-3-phosphate dehydrogenase, or from Gallus gallus β-actin, but are not limited thereto.

[0149] In some embodiments, the control sequence is a suitable transcription terminator sequence that is a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that functions in the selected host cell can be used in the present invention. For example, exemplary transcription 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 of 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, e.g., Romanos et al., supra). Exemplary terminators for mammalian cells include those from cytomegalovirus (CMV), simian vacuolating virus 40 (SV40), Homo sapiens growth hormone hGH, those from bovine growth hormone BGH, and those from human or rabbit beta-globin, but are not limited thereto.

[0150] In some embodiments, the control sequences are suitable leader sequences, 5' cap modifications, 5' UTRs, etc. 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 linked to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that functions in the 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 obtained from the genes of Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP). Suitable leaders for mammalian host cells include, but are not limited to, the 5'-UTR element present in orthopoxvirus mRNA.

[0151] In some embodiments, the control array is an array operably linked to the 3' end of the protein-coding nucleic acid array, and is an array that mediates binding to proteins involved in mRNA transport and translation and mRNA half-life, and includes a 3' untranslated nucleic acid region and a polyadenylation tail nucleic acid array. Any polyadenylation sequence and 3' UTR that function in the selected host cell can be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, those from the genes of 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] ). Mam Useful polyadenylation and 3' UTR sequences for mammalian host cells include, but are not limited to, the 3'-UTRs of alpha- and beta-globin mRNAs, which carry several sequence elements that improve mRNA stability and increase translation.

[0152] In some embodiments, the control array is a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the secretory pathway of the cell. The 5' end of the coding sequence of the nucleic acid sequence may originally contain a signal peptide coding region that is naturally linked in-frame with 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 that is heterologous to the coding sequence. Any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of the selected host cell is used in the expression of the engineered GAA polypeptide provided herein. Effective signal peptide coding regions for bacterial host cells include, but are not limited to, signal peptide coding regions obtained from the genes of Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are known in the art (see, for example, Simonen and Palva, Microbiol. Rev., 57:109-137

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

[0153] In some embodiments, the control sequence is a propeptide coding region encoding an amino acid sequence located at the amino terminus of the polypeptide. The resulting polypeptide is in some cases referred to as a "proenzyme", "propolypeptide" or "zymogen". By catalytic or autocatalytic cleavage of the propeptide from the propolypeptide, the propolypeptide can be converted into a mature active polypeptide. The propeptide coding region includes, but is not limited to, the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (see, for example, WO95 / 33836). When both a signal peptide region and a propeptide region are present 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 the expression of the polypeptide in relation to the growth of the host cell. Examples of regulatory systems are those that turn gene expression on or off in response to chemical or physical stimuli, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to, the lac, tac and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to, the ADH2 system or the GAL1 system. In filamentous fungi, suitable 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 aspect, 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 a promoter, a terminator, an origin of replication, etc., depending on the type of host to be introduced. In some embodiments, the various nucleic acids and control sequences described above are joined to each other to generate a recombinant expression vector that contains one or more convenient restriction sites to allow insertion or substitution of a nucleic acid sequence encoding a 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 containing the polynucleotide sequence, into an appropriate vector for expression. When constructing an expression vector, the coding sequence is positioned within the vector such that the coding sequence is operably linked to appropriate control sequences for expression.

[0156] The recombinant expression vector can be conveniently subjected to recombinant DNA procedures, and as a result, can cause the expression of a variant GAA polynucleotide sequence, and can be any suitable vector (e.g., an adenovirus (AV), an adeno-associated virus (AAV), a lentivirus (LV), and non-viral vectors, such as plasmids or viruses, including but not limited to liposomes). The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear plasmid or a closed circular plasmid. In fact, it is not intended that the present invention 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, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may contain some means for ensuring self-replication. In some alternative embodiments, the vector may be one that integrates into the genome when introduced into the host cell and is replicated along with the chromosome into which it has integrated. Further, a single vector or plasmid may be used, or two or more vectors or plasmids containing all the DNA that will be introduced into the host cell's genome or transposon when combined may be used.

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

[0159] In some embodiments, the expression vector preferably contains one or more selectable markers that enable easy selection of transformed cells. A "selectable marker" is a gene whose product confers biocide or virus resistance, resistance to heavy metals, prototrophy to auxotrophic strains, and the like. Examples of selectable markers for bacteria include the dal gene from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance, but are not limited thereto. 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 amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenylyltransferase), and trpC (anthranilate synthase), and equivalents thereof, but are not limited thereto. In another aspect, 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 control sequences for expression of the engineered GAA enzyme in the host cell. Host cells for use in expressing the polypeptide encoded by the expression vector of the present invention are well known in the art and include bacterial cells such as E. coli, Vibrio fluvialis, Streptomyces, and Salmonella typhimurium cells, fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris [ATCC accession number 201178]), insect cells, such as 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, Jurkat, and Bowes melanoma cell lines), and plant cells, including but not limited to these.

[0160] Accordingly, in another aspect, the present invention provides a method for producing an engineered GAA polypeptide, the method comprising 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 isolating and / or purifying the GAA polypeptide as described herein.

[0161] Suitable culture media and growth conditions for the above host cells are well known in the art. The polynucleotide for GAA polypeptide expression can be introduced into cells by various methods known in the art. Examples of techniques include, among others, electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0162] An engineered GAA having the characteristics disclosed herein can be obtained by subjecting a polynucleotide encoding a naturally occurring or engineered GAA polypeptide to mutagenesis and / or directed evolution methods known in the art and described herein. Exemplary directed evolution techniques are 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 evolution procedures that can be used include, among others, the staggered extension 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 mutagenesis (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 readily 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. Patent Nos. 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; 6,287,861; 6,287,862; 6,291,242; 6,297,053; 6,303,344; 6,309,883; 6,319,713; 6,319,714; 6,323,030; 6,326,204; 6,335,160; 6,335,198; 6,344,356; 6,352,859; 6,355,484; 6,358,740; 6,358,742; 6,365,377; 6,365,408; 6,368,861; 6,372,497; 6,337,186; 6,376,246; 6,379,964; 6,387,702; 6,391,552; 6,391,640; 6,395,547; 6,406,855; 6,406,910; 6,413,745; 6,413,774; 6,420,175; 6,423,542; 6,426,224; 6,436,675; 6,444,468; 6,455,253; 6,479,652; 6,482,647; 6,483,011; 6,484,105; 6,489,146; 6,500,617; 6,500,639; 6,506,602; 6,506,603; 6,518,065; 6,519,065; 6,521,453; 6,528,311; 6,537,746; 6,573,No. 098, No. 6,576,467, No. 6,579,678, No. 6,586,182, No. 6,602,986, No. 6,605,430, No. 6,613,514, No. 6,653,072, No. 6,686,515, No. 6,703,240, No. 6,716,631, No. 6,825,001, No. 6,902,922, No. 6,917,882, No. 6,946,296, No. 6,961,664, No. 6,995,017, No. 7,024,312, No. 7,058,515, No. 7,105,297, No. 7,148,054, No. 7,220,566, 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, 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, No. 8,048,674, No. 8,058,001, No. 8,076,138, No. 8,108,150, No. 8,170,806, No. 8,224,580, No. 8,377,681, No. 8,383,346, No. 8,457,903, No. 8,504,498, No. 8,589,085, No. 8,762,066, No. 8,768,871, No. 9,593,326, No. 9,684,771, No. 9,665,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, as well as all related US and non-US 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., 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. Op. 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. Nat. Acad. Sci. U.S.A., 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] are hereby incorporated by reference in their entirety.,

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

[0165] For engineered polypeptides of known sequence, polynucleotides encoding the enzyme can be prepared by standard solid-phase methods according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be synthesized individually and then joined (e.g., by enzymatic or chemical ligation methods, or methods mediated by polymerase) to form any desired contiguous sequence. For example, the polynucleotides and oligonucleotides disclosed herein are usually performed by automated synthesis methods, but can be prepared by chemical synthesis using classical phosphoramidite methods (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., on an automated DNA synthesizer), purified, annealed, ligated, and cloned into an appropriate vector.

[0166] Thus, in some embodiments, a method for preparing an engineered GAA polypeptide can include: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the amino acid sequences 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, and SEQ ID NO: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104, and any variants thereof; and (b) expressing the GAA polypeptide encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide can have one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions, and / or substitutions, as necessary. In some embodiments, the amino acid sequence can have 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, as necessary. In some embodiments, the amino acid sequence can have 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, as necessary. In some embodiments, the amino acid sequence can have 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, as necessary. In some embodiments, the substitutions can be conservative substitutions or non-conservative substitutions.

[0167] The expressed and engineered 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, any, some, or all of the engineered GAA polypeptides expressed in the host cell are recovered from the cells and / or the culture medium using one or more of the well-known techniques for protein purification, including but not limited to, among others, lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography.

[0169] Chromatography techniques for the isolation of the GAA polypeptide 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 will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., 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 the production of antibodies, various host animals including but not limited to rabbits, mice, rats, etc. are immunized by injection with the GAA polypeptide (e.g., GAA variant) or a fragment thereof. In some embodiments, the GAA polypeptide or fragment is conjugated to a suitable carrier such as BSA by side chain functional groups or by a linker attached to the side chain functional groups.

[0170] In some embodiments, the engineered GAA polypeptide is produced in a host cell by a method comprising culturing a host cell (e.g., S. cerevisiae, Daucus carota, Nicotiana tabacum, H. sapiens [e.g., HEK293T], or Cricetulus sp. [e.g., CHO]) comprising 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 the culture medium.

[0171] In some embodiments, the engineered GAA polypeptide is produced in a host cell by a method comprising culturing a host cell comprising 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 the culture medium.

[0172] In some preferred embodiments, the present invention provides a method for producing an engineered GAA polypeptide, comprising culturing a recombinant bacterial cell comprising a polynucleotide sequence encoding an engineered GAA polypeptide that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to reference sequences SEQ ID NO: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104 when aligned, and has one or more amino acid residue differences compared to SEQ ID NO: 2, 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, and / or 3104, and / or combinations thereof, under culture conditions suitable for enabling the production of the engineered GAA polypeptide, and optionally, recovering the engineered GAA polypeptide from the culture and / or the cultured bacterial cells.

[0173] In some embodiments, the engineered GAA polypeptide, when recovered from a recombinant host cell or cell culture, 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 include the engineered GAA polypeptide for different applications and uses (e.g., pharmaceutical compositions). Composition:

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

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

[0176] In some embodiments, the engineered GAA polypeptide is formulated for use in an oral pharmaceutical composition. Any form suitable for use in delivering the engineered GAA polypeptide can be used in the present invention, and such forms include, but are not limited to, pills, tablets, gel tabs, capsules, troches, dragees, powders, soft gels, sol-gels, gels, emulsions, implants, patches, sprays, ointments, liniments, creams, pastes, jellies, topical applications, aerosols, chewing gums, lubricants, sticks, suspensions (including but not limited to oil suspensions, water-in-oil emulsions, etc.), slurries, syrups, controlled release formulations, suppositories, and the like. In some embodiments, the engineered GAA polypeptide is provided in a form suitable for injection (i.e., an injectable formulation). In some embodiments, the engineered GAA polypeptide is provided in a biocompatible matrix such as a sol-gel including, for example, a silica-based (e.g.,oxysilane) sol-gel. In some embodiments, the engineered GAA polypeptide is encapsulated. In some alternative embodiments, the engineered GAA polypeptide is encapsulated in nanostructures (e.g., nanotubes, nanowires, nanocapsules, or microcapsules, microspheres, liposomes, etc.). In fact, it is not intended that the present invention 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, via implant, and the like.

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

[2005] ; U.S. Patent Nos. 7,531,341, 7,534,595, 7,5 (See, e.g., U.S. Patent Nos. 60,263 and 7,536,53, U.S. Patent Application Publication Nos. 2013 / 0039898 and 2012 / 0177722, etc.). In fact, 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 such as 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 the polynucleotide sequence in encapsulated delivery such as liposomes. In some additional embodiments, the engineered GAA polypeptide is provided for delivery to cells or tissues by cell therapy, wherein a polynucleotide sequence encoding the engineered GAA polypeptide is introduced into exogenous cells, and the cells (or cell) are introduced into a recipient (e.g., a patient presenting 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 cross-linked crystalline engineered GAA enzyme and a polymer having a reactive moiety attached to the enzyme crystal. The present invention also provides an engineered GAA polypeptide in the polymer.

[0180] In some embodiments, a composition comprising an engineered GAA polypeptide of the invention comprises 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), celluloses (e.g., methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose), gums (e.g., gum arabic, tragacanth gum, guar gum, etc.), and / or proteins (e.g., gelatin, collagen, etc.). Additional components in oral formulations can include coloring and / or sweetening agents (e.g., glucose, sucrose, and mannitol) and lubricants (e.g., magnesium stearate), as well as enteric coating agents (e.g., methacrylate polymers, hydroxypropylmethylcellulose phthalate, and / or any other suitable enteric coating agent known in the art). In some embodiments, a disintegrating or solubilizing agent is included (e.g., crosslinked polyvinylpyrrolidone, agar, alginic acid or its salts, such as sodium alginate). In some embodiments, the engineered GAA polypeptide is combined with various additional components including, but not limited to, preservatives, suspending agents, thickening agents, wetting agents, alcohols, fatty acids, and / or emulsifying agents, particularly in liquid formulations. In some embodiments, the engineered 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, and 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 gelcaps, further having an enteric coating.

[0181] In some embodiments, the present invention provides an engineered GAA polypeptide suitable for use in reducing the concentration of glycogen in a tissue. The dosage of the engineered GAA polypeptide administered to an animal depends on the disease state, the general condition of the animal, and other factors known to those of skill in the art. In some embodiments, the composition is for single or multiple administrations to an animal. In some embodiments, the concentration of the engineered GAA polypeptide in the composition administered to an animal (e.g., a human having Pompe disease) is intended to be sufficient to effectively treat, ameliorate, 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, the engineered GAA polypeptide is administered in combination with other pharmaceutical and / or dietary compositions.

[0182] The above and other aspects of the present invention can be better understood in connection with the following non-limiting examples. These examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

Examples

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

[0184] In the experiments disclosed below, the following abbreviations apply: ppm (parts per million); M (molarity); mM (millimolarity); uM and μM (micromolarity); nM (nanomolarity); mol (mole); gm and g (gram); mg (milligram); ug and μg (microgram); L and l (liter); ml and mL (milliliter); cm (centimeter); mm (millimeter); um and μm (micrometer); sec. (second); min (minute); h and hr (hour); U (unit); MW (molecular weight); rpm (revolutions per minute); °C (degrees Celsius); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coli W3110 (a commonly used laboratory E. coli strain available from the Coli Genetic Stock Center [CGSC], New Haven, CT); DPBS (Dulbecco's 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 performance liquid chromatography); SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis); MU-Glu (4-methylumbelliferyl α-D-glucopyranoside); IPTG (isopropyl β-D-1-thiogalactopyranoside); PMBS (polymyxin B sulfate); FIOPC (fold improvement over positive control); PBMC (peripheral blood mononuclear cell); 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) Obtaining the GAA gene and constructing an expression vector

[0185] In this example, obtaining the GAA gene and constructing an expression vector are described. 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 the yeast MFα signal peptide sequence (SEQ ID NO: 3383) to generate the gene sequence represented by SEQ ID NO: 2, which was cloned into the yeast expression vector pYT-72 as previously described (see, for example, US Patent Application Publication No. 2017 / 0360900A1). Recombinant cloning and gene expression were performed in the S. cerevisiae strain INVSc1. A library of gene variants was generated from this plasmid construct using directed evolution techniques (see, for example, US Patent No. 8,383,346 and WO2010 / 144103).

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

[0187] The pDH vector was generated such that the vector copy number, the desired selection drug, and the base pair length were optimized to enable compatibility with library variant generation. For mammalian expression, the PCR product was ligated into either the BamHI / XhoI site or the HindIII / XhoI site of the linearized pDH vector. Using directed evolution, specific gene variants (SEQ ID NOs: 829 - 3378) derived from SEQ ID NO: 20 were generated within the pDH plasmid construct. The plasmid map for pDH containing WT GAA is shown in FIG. 9, and the plasmid sequence is provided as SEQ ID NO: 3379. Additionally, the plasmid map for pDH containing the beta - lactamase (bla) stuffer sequence is provided in FIG. 10, and the sequence is provided as SEQ ID NO: 3380.

[0188] In some experiments, the expression of GAA variants was performed using the linear PCR amplification products of the above - mentioned expression cassettes in pcDNA3.1(+) or pDH (i.e., chimeric expression constructs composed of a synthetic mouse IG signal peptide (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 and 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] In this example, experiments involving high - throughput growth of cells producing GAA variants and assays for determining GAA activity are described. High - throughput (HTP) growth of S. cerevisiae

[0190] Yeast (INVSc1) cells transformed with vectors expressing GAA and GAA variants using the lithium acetate method known in the art were selected on SD-Ura agar plates. After incubation at 30 °C for 72 h, colonies were placed into wells of an AXYGEN® 1.1 ml 96-well deep well plate (Axygen) filled with 200 μl / well of SD-Ura broth supplemented with 6% glucose (2 g / L SD-Ura, 6.8 g / L yeast nitrogen base without amino acids [Sigma Aldrich]), 3.06 g / L sodium dihydrogen phosphate, 0.804 g / L disodium hydrogen phosphate, pH 6.0. The cells were grown for 20 - 24 h in a Kuhner shaker (250 rpm, 30 °C, and 85% relative humidity). An 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 in a Kuhner shaker (250 rpm, 30 °C, and 85% relative humidity) for 66 - 84 h. The cells were then pelleted (4000 rpm × 20 min) and the supernatant (conditioned 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-exposed assay, 50 μL of the SD-URA conditioned medium generated 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 mixture was briefly mixed and incubated at 37 °C for 4 - 24 h, 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 the supernatant exposed to neutral buffer

[0192] The GAA variant was exposed to a neutral to near-neutral (pH 6.5 - 7.5) buffer to simulate the pH that the variant would encounter in the blood after administration to a patient. First, 50 μL of the GAA variant in SD-URA conditioned medium and 50 μL of McIlvaine 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. Then, 50 μL of each exposed sample was mixed with 50 μL of 1 mM 4-MUGlu in McIlvaine buffer, pH 4.4. The reaction mixture was mixed briefly 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 the supernatant pretreated with acid

[0193] The GAA variant was exposed to an acidic (pH 3) buffer to simulate the pH that the variant would encounter in lysosomes after administration to a patient. First, 50 μL of the GAA variant in SD-URA conditioned medium and 50 μL of McIlvaine 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. Then, 50 μL of each exposed sample was mixed with 50 μL of 1 mM 4-MUGlu in McIlvaine buffer, pH 4.4. The reaction mixture was mixed briefly 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). HTP analysis of the supernatant with glycogen substrate

[0194] The hydrolytic activity of the GAA variant was determined by measuring the hydrolysis of glycogen to glucose. For the non-exposed assay, 50 μL of the SD-URA conditioned medium generated as described above was mixed with 50 μL of 5 mM glycogen in pH 4.5 McIlvaine buffer (7.71 mL of 0.2 M Na 2 HPO 4 and 12.29 of 0.1 M citric acid) in a 96-well, black, opaque-bottom plate. The reaction mixture was mixed briefly and incubated at 37 °C for 24 h. After incubation, 20 μL of the glycogen hydrolysis reaction mixture was mixed with 80 μL of the AMPLEX® Red Glucose Assay Kit (Sigma) to determine the glucose content of each reaction. 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 activities were determined by assaying enzyme activity after a series of independent exposures. Results for all of the variants and the substitutions in each of their polypeptide sequences are reported in relation to SEQ ID NO: 2. These variants were tested for GAA 4-MUGlu activity (FIOPC of non-exposed activity), after pH 6.5 incubation (FIOPC of pH 6.5 stability activity), and for glycogen hydrolysis (FIOPC of 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

[0196] In this example, the analysis of the GAA variant derived from SEQ ID NO: 8 for the improvement of GAA activity after a series of exposures will be described. The directed evolution of GAA encoded by SEQ ID NO: 8 was performed by constructing a library of variant genes. These libraries were then seeded, grown, and screened for GAA 4-MUGlu activity in a non-exposed, no pre-incubation activity assay (FIOPC of non-exposed activity), after incubation at pH 7 (FIOPC of pH 7 stability activity), or after incubation at pH 3 (FIOPC of pH 3 stability activity), as described in Example 2. The results are presented in Table 4-1.

Table 4-1-1

Table 4-1-2

Table 4-1-3

[0197] The pcDNA 3.1(+) vector (ThermoFisher Scientific), pDH vector, or PCR-amplified linear DNA (as described in Example 1) encoding the synthetic mouse Ig signal peptide (SEQ ID NOs: 3381 and 3382) fused to wild-type GAA or a GAA variant was transfected into HEK 293T cells using the lipofection method with LIPOFECTAMINE® 3000 reagent (ThermoFisher Scientific). HEK 293T cells were cultured in standard complete growth medium (DMEM containing 10% fetal bovine serum, both from Corning) and seeded at a density of 0.5×10 5 cells / well / 250 μL in NUNC® Edge 2.0 96-well plates (ThermoFisher Scientific) and allowed to adhere and grow for 24 hours at 37°C in 5% CO 2 2. After that, they were subjected to lipofection-mediated transfection. After transfection, the cells were incubated for 24 - 96 hours to allow expression of the GAA variant and secretion into the conditioned medium. Then, the conditioned medium (20 - 100 μL) from the HEK293T transfection was 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 unexposed assay, 20 μL of the HEK 293T conditioned medium generated 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 reactants were incubated at 25 - 37 °C for 30 - 60 minutes while 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 (Molecular Devices) or an ENVISION® microplate reader (Perkin Elmer). HTP analysis of supernatant exposed to neutral buffer

[0199] GAA variants were exposed to neutral buffer to simulate the pH that the variants would encounter in blood after administration to patients. First, 20 μL of conditioned medium containing GAA variants 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 Mcllvaine buffer, pH 4.4. The reactants were incubated at 37 °C for 180 minutes while 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] The GAA variants were exposed to plasma to simulate the conditions the variants would encounter in the blood after administration to a patient. First, 30 μL of conditioned medium containing the GAA variants 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-exposed sample was mixed with 50 μL of 1.5 mM 4-MUGlu in McIlvaine buffer, pH 4.4. The reaction was incubated at 25 - 37 °C for 15 - 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 or an ENVISION® microplate reader (Perkin Elmer). HTP analysis of GAA activity in lysates of pump fibroblasts and C2C12 GAA knockout myoblasts

[0201] The GAA variants produced by HTP were incubated with target cells, and the residual intracellular activity was assayed 24 hours later. For these experiments, mammalian cells lacking functional GAA activity, namely, fibroblasts from Pompe patients (Coriell Institute for Medical Research #GM00248), and C2C12 myoblasts in which the native GAA gene was knocked out using Crispr-Cas9 editing were used. In these experiments, Pompe fibroblasts or C2C12 GAA knockout myoblasts were seeded in black, clear-bottom, tissue culture-treated COSTAR® 96-well plates (Corning, 3904) and allowed to grow to confluence in standard complete growth medium. Once confluent, the complete growth culture medium was removed from the plates using an automated BIOMEK® i5 liquid handling robot. Conditioned medium produced by transiently transfected HEK293T cells as described above was transfected into fibroblasts from Pompe patients and C2C12 myoblasts and incubated at 37 °C, 5% CO 2 for 4 - 24 hours. The medium was removed from the cultures using an automated BIOMEK® i5 liquid handling robot. The cells were washed briefly with 150 μL of 1× DPBS / well, and the DPBS was removed using an automated BIOMEK® i5 liquid handling robot. Then, 200 μL of standard complete growth culture medium was added to each well, and the plates were returned to the incubator for 0 - 72 hours. At the end of the incubation, the standard complete growth medium was removed using an automated BIOMEK® i5 liquid handling robot. The cells were washed with 150 μL of 1× DPBS / well, and the DPBS was removed using automated BIOMEK® i5 liquid handling. 0.2 - 0.5% TRITON X-100™ nonionic surfactant (Sigma Cells were lysed by supplementing 50 μL of McIlvaine buffer, pH 4.4, adding <#93443>, and stirring for 30 minutes at room temperature. Activity was assayed by adding 50 μL of 1.5 mM 4-MUGlu in McIlvaine 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 or an ENVISION® microplate reader (Perkin Elmer). FIOPC of cell uptake was calculated by dividing the intracellular activity of the GAA variant by the activity of the reference polypeptide having the indicated SEQ ID NO. (Example 6) GAA variant of SEQ ID NO:16

[0202] In this example, the evolution of GAA variants 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 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 unexposed activity”) as described in Example 5 and also after pH 7.4 pre-incubation (“FIOPC of pH 7.4 stability and activity”). Variants were also tested for 4-MUGlu activity after lysis of Pompe fibroblasts (“FIOPC of activity from Pompe fibroblast lysate”) or GAA - / - after lysis of C2C12 cells (“FIOPC of activity from C2C12 GAA - / - lysate”). The results of these assays are presented in Table 6-1.

Table 6-1-1

Table 6-1-2

Table 6-1-3

Table 6-1-4

Table 6-1-5

[0203] In this example, the generation of GAA variants is described. Production of GAA in EXPI293F (trademark) Cells

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

[0205] In this example, a method for purifying GAA variants is described. Purification of GAA Variants from Mammalian Cell Supernatants

[0206] The 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 as described in Example 7 were purified from mammalian culture supernatants 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 MgCl 2 , CaCl 2 , and MnCl 2 , pH 6.0 (Concanavalin A binding buffer). The supernatant was sterile filtered through a 0.2 μm bottle top filter and then loaded onto the column. After loading, the column was washed with 10 column volumes of Concanavalin A binding buffer, and the bound protein was eluted with Concanavalin A binding buffer supplemented with 0.9 M methyl-α-D-mannopyranoside and 0.9 M methyl-α-D-glucopyranoside. The eluted protein was concentrated and buffer exchanged into 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 molecular weight cut-off membrane (Millipore). The GAA in the storage buffer was sterile filtered through an ANOTOP® 0.2 μm syringe filter (Whatman) and stored at -80°C. Based on BCA quantification (described below), 2.4 - 50 ng of purified protein per ml of culture supernatant was obtained by this purification process. 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 used in 96-well, black, opaque-bottom plates with 50 μL of 0 to 2.5 mM 4-MUGlu in McIlvaine buffer (McIlvaine, J. Biol. Chem., 49:183-186

[1921] ), pH 4.5. The reaction mixture 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 variants against neutral pH was determined by incubating the variants 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 McIlvaine buffer, pH 4.4, in a 96-well, black, opaque-bottom plate. After incubating the reaction mixture with stirring for 30 minutes at room temperature, 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 measured by monitoring fluorescence (Ex. 355 nm, Em. 460 nm) using a SPECTRAMAX® M2 microplate reader or an ENVISION® microplate reader (Perkin Elmer), and the hydrolysis was analyzed. The results from this assay are presented in FIG. 11. Cell uptake of purified GAA variants expressed in Expi293F cells into Pompe fibroblasts or C2C12 GAA knockout myoblasts

[0211] Cell uptake of the GAA variants was determined by comparison to a reference enzyme (SEQ ID NOs: 4 and 6) to assess their overall ability to be endocytosed by cultured cells. Pompe fibroblasts (GM00248, Coriell Institute for Medical Research) or C2C12 GAA knockout cells were seeded in standard complete growth medium in a black-wall, clear-bottom 96-well plate (Costar, #3603) and allowed to become confluent (37 °C, 5% CO 2 for 2-3 days). After becoming confluent, the standard complete growth medium was removed using an automated BIOMEK® i5 liquid handling robot. The enzyme purified as described in Example 8 was added to the cells at 0-10 μg GAA / mL in standard complete growth medium and incubated at 37 °C, 5% CO 2It was left to incubate. Using an automated BIOMEK® i5 liquid handling robot, the medium containing the GAA variant was aspirated. The cells were washed briefly with 150 μL of 1× DPBS / well, and the DPBS was removed by the automated BIOMEK® i5 liquid handling robot. Then, 200 μL of standard complete growth medium was added to each well, and the plate was returned to the incubator for 0 - 72 hours. At the end of incubation, the MEM complete growth medium was removed using the automated BIOMEK® i5 liquid handling robot. The cells were washed with 150 μL of 1× DPBS / well, and the DPBS was removed using the automated BIOMEK® i5 liquid handling robot. The cells were lysed by the addition of 50 μL of McIlvaine buffer, pH 4.4, supplemented with 0.2 - 0.5% TRITON X-100™ non-ionic surfactant (Sigma #93443), and stirring at room temperature for 30 minutes. GAA activity was assayed by the addition of 50 μL of 1.5 mM 4-MUGlu in McIlvaine buffer, pH 4.4. The plate was 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. The FIOPC of cellular uptake was calculated by dividing the intracellular activity of the GAA variant by the control (WT) activity normalized. Figures 4, 5, 8, 12, 13, 16 - 18, 22, 24, and 26 provide graphs showing the cellular uptake of purified GAA variants into pump 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 a graph showing the cellular uptake and stability of the GAA variant after 1 - 7 days. Glycogenolytic activity of GAA variants expressed in EXPI293F™ cells

[0212] The relative glycogenolytic activity of the GAA variants was evaluated using the AMPLEX® Red Glucose / Glucose Oxidase Kit (Invitrogen, #A22189) according to the manufacturer's instructions with the modifications described herein. Briefly, the GAA variants were diluted to an appropriate concentration range (0 - 2 μg / mL) for a 50 μL reaction in a COSTAR® black-wall, clear-bottom 96-well plate (#3603, Corning) with 10 mM glycogen in McIlvaine buffer, pH 4.4. The plate was sealed and gently shaken at room temperature for 30 minutes. The reaction was neutralized by the addition of 25 μL of 0.5 M sodium carbonate, pH 10.5. 50 μL of the AMPLEX® Red / horseradish peroxidase / glucose oxidase solution prepared according to the manufacturer's instructions was added to each well. The plate was sealed and gently shaken at room temperature for 30 minutes. Fluorescence (Ex. 530 nm, Em. 590 nm) was monitored using a SPECTRAMAX® M2 microplate reader to quantify the reaction against a glucose-free standard curve. The results are shown in Figure 2. (Example 10) GAA variant of SEQ ID NO: 20

[0213] In this example, experiments on the evolution of the GAA variant derived from SEQ ID NO: 20 and screening for improvement of GAA activity after a series of exposures are described. A library of variant genes GAA encoded by SEQ ID NO: 20 / based on SEQ ID NO: 20 was constructed, seeded, grown, screened for GAA MU-Glu activity ("FIOPC of unexposed activity"), and also screened after plasma exposure ("FIOPC of plasma stability and activity") as described in Example 5. The variants were also screened for 4-MuGlu activity as described in Example 5, after lysis of Pompe fibroblasts ("FIOPC of activity from Pompe fibroblast lysate") or GAA - / - after lysis of C2C12 cells ("C2C12 GAA - / -Tested for "FIOPC of activity from dissolved substances". 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 a synthetic mouse IG signal peptide (SEQ ID NOs: 3381 and 3382) fused to wild-type GAA or a GAA variant was transfected into EXPI293F™ cells (ThermoFisher Scientific) using the 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 a density of 1×10 6 cells / well / 400 μL in Axygen 1.1 mL deep well plates (Corning, P-DW-11-C-S). The cells were subjected to lipofection-mediated transfection and returned to a shaking incubator with 8% CO 2 and 70% humidity for 3 - 4 days to allow expression of the GAA variant and secretion into the conditioned medium. The conditioned medium was collected by centrifugation of the expression plates and transfer of the conditioned medium to BioRad Hardshell PCR Plates (BioRad, HSP9601). The plates were centrifuged again and the clarified conditioned medium was transferred to new 96-well plates for analysis of activity, stability or uptake into cells. HTP analysis of the supernatant

[0215] The GAA variant activity was determined by measuring the hydrolysis of 4-methylumbelliferyl α-D-glucopyranoside (4-MUGlu). For the non-exposed assay, 5–10 μL of the clarified conditioned medium of EXPI293F™ generated 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 min 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). The FIOPC of the non-exposed activity was calculated by dividing the GAA variant by the activity of the reference polypeptide with the indicated SEQ ID NO. HTP analysis of the supernatant exposed to plasma

[0216] The GAA variants were exposed to plasma to simulate the conditions the variants would encounter in the blood after administration to patients. First, 30 μL of the GAA variant in EXPI293F™ clarified conditioned medium was combined with 30 μL of plasma (Innovative Research, Innovative Grade US Origin Monkey Cynomolgus Plasma K2 EDTA) in a 96-well plate. The plate was sealed and incubated at 37 °C for 2 - 4 hours. Next, 10 μL of the plasma-expo...

Claims

[Claim 1] The invention described in the specification.