Engineered acid alpha-glucosidase variants

Abstract

The present disclosure provides engineered acidic alpha-glucosidase polypeptides, recombinant polynucleotides encoding the engineered acidic alpha-glucosidase polypeptides, and methods of using the engineered acidic alpha-glucosidase polypeptides and the recombinant polynucleotides for therapeutic purposes.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480044624.5 (22) Application Date 2024.06.28 (30) Priority Data 63 / 511,347 2023.06.30 US (85) PCT International Application Entering National Phase Date 2025.12.30 (86) PCT International Application Application Data PCT / US2024 / 036223 2024.06.28 (87) PCT International Application Publication Data WO2025 / 007035 EN 2025.01.02 (71) Applicant: Crosswalk Therapeutics, Inc. Address: Massachusetts, USA (72) Inventors: Rachel Kathleen Botham, Gordon Michael Zhang, William Casey Harros, Giggart W. Hasman, Nikki D. Cruz, Jessica P. Rauschpa Maroju, Adam P. Silverman (74) Patent Agency: Beijing Anxin Fangda Intellectual Property Agency Co., Ltd. 11262 Patent Attorneys: Wang Weiwei, Zheng Xia (51) Int.Cl. C12N 9 / 26 (2006.01) A61K 38 / 47 (2006.01) A61P 3 / 00 (2006.01) (54) Invention Title: Engineered Acidic α-Glucosidase Variants (57) Abstract: This disclosure provides engineered acidic α-glucosidase polypeptides, recombinant polynucleotides encoding said engineered acidic α-glucosidase polypeptides, and the engineered acidic α-glucosidase polypeptides... Method for using the α-glucosidase polypeptide and the recombinant polynucleotide for therapeutic purposes. Claims 3 pages, Description 115 pages, Sequence Listing (electronic publication), Drawings 8 pages. CN 121511299 A 2026.02.10 CN 1 21 51 12 99 A 1. An engineered acidic α-glucosidase or a bioactive fragment thereof, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 12, wherein the amino acid sequence comprises at least substituted or amino acid residues 305V, 24A / C / D / F / G / H / I / K / M / N / P / S / T / V / Y, 28A / C / D / E / F / G / H / K / Q / T / V / W, 29A / C / D / E / F / G / H / I / K / M / N / P / R / W / Y, 39A / E / F / G / I / L / N / T, 50A / C / D / E / F / H / I / K / M / N / R / S / T / W / Y, 62D / H / I / K / M / N / P / Q / Y, 78A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 87A / G / H / I / K / L / MN / Q / R / S / T / V / W, 135C / D / E / F / G / H / I / K / L / N / R / Y, 266A / D / E / H / K / Q, 267H / L / T / V, 437A / H, 486C / D / F / G / H / I / K / L / M / N / Q / R / S / V / W / Y, 522A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y, 569A / C / D / E / G / K / M / N / P / R / W, 670A / D / G / H / K / M / Y, 692A / D / E / H / K / L / M / N / T / W, 711D / E / I / K / M / N / Q / S / T / V / Y, 736F / L, 750E / K / L / Q / R, 812A / D / G / S, 830D / E / F / G / H / L / M / N / S / T / W / Y, 842A / C / D / F / H / K / L / M / N / Q / R / T / W, 871A / C / D / F / H / I / M / N / Q / T / V / W / Y, 883A / F / Q, 894A / D / E / H / I / K / L / M / N / S / T / V / W / Y, 913F / I / K / M / N / S or 932C / D / E / G / H / K / L / M / N / P / Q / R / W / Y, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2, or relative to the reference sequence corresponding to SEQ ID NO: 12 or 2. 2. An engineered acidic α-glucosidase or a bioactive fragment thereof, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with the reference sequence of the even-numbered SEQ ID NOs in SEQ ID NOs: 14 to 754, wherein said amino acid sequence comprises at least substituted or amino acid residues 305V, 24A / C / D / F / G / H / I / K / M / N / P / S / T / V / Y, 28A / C / D / E / F / G / H / K / Q / T / V / W, 29A / C / D / E / F / G / H / I / K / M / N / P / R / W / Y, 39A / E / F / G / I / L / N / T, 50A / C / D / E / F / H / I / K / M / N / R / S / T / W / Y, 62D / H / I / K / M / N / P / Q / Y, 78A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 87A / G / H / I / K / L / MN / Q / R / S / T / V / W, 135C / D / E / F / G / H / I / K / L / N / R / Y, 266A / D / E / H / K / Q, 267H / L / T / V, 437A / H, 486C / D / F / G / H / I / K / L / M / N / Q / R / S / V / W / Y, 522A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y, 569A / C / D / E / G / K / M / N / P / R / W, 670A / D / G / H / K / M / Y, 692A / D / E / H / K / L / M / N / T / W, 711D / E / I / K / M / N / Q / S / T / V / Y, 736F / L, 750E / K / L / Q / R, 812A / D / G / S, 830D / E / F / G / H / L / M / N / S / T / W / Y, 842A / C / D / F / H / K / L / M / N / Q / R / T / W, 871A / C / D / F / H / I / M / N / Q / T / V / W / Y, 883A / F / Q, 894A / D / E / H / I / K / L / M / N / S / T / V / W / Y, 913F / I / K / M / N / S or 932C / D / E / G / H / K / L / M / N / P / Q / R / W / Y, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2. 3. The engineered acidic α-glucosidase according to claim 1 or 2, wherein the amino acid sequence of the engineered acidic α-glucosidase comprises residues 20 to 944 of SEQ ID NO. with even numbers from SEQ ID NO: 14 to 754, or comprises SEQ ID NO. with even numbers from SEQ ID NO: 14 to 754. 4. The engineered acidic α-glucosidase according to claim 1 or 2, wherein the amino acid sequence of the engineered acidic α-glucosidase comprises residues 20 to 944 of SEQ ID NO: 14, 114, 126, 170, 250, 252, 394, 472, 488, or 506, or comprises SEQ ID NO: 14, 114, 126, 170, 250, 252, 394, 472, 488, or 506.5. The engineered acidic α-glucosidase according to any one of claims 1 to 4, wherein, compared with a reference acidic α-glucosidase having a sequence of residues 20 to 944 corresponding to SEQ ID NO: 2 or 12 or a sequence corresponding to SEQ ID NO: 2 or 12, the engineered acidic α-glucosidase exhibits at least one of the following modified properties selected from: i) enhanced catalytic activity; ii) improved tolerance to pH 7; iii) improved tolerance to pH 4.4; iv) improved stability in lysosomes; v) improved expression in cells; vi) improved uptake into cells; vii) improved enzymatic activity in cell lysates; viii) improved stability in plasma / serum; and ix) reduced immunogenicity; or a combination of any one of i), ii), iii), iv), v), vi), vii), viii), and ix). 6. The engineered acidic α-glucosidase according to claim 5, wherein the engineered acidic α-glucosidase exhibits reduced immunogenicity compared to a reference acidic α-glucosidase having a sequence corresponding to residues 20 to 944 of SEQ ID NO: 2 or 12 or a sequence corresponding to SEQ ID NO: 2 or 12. 7. The engineered acidic α-glucosidase according to claim 6, wherein the engineered acidic α-glucosidase exhibits the following: (a) a total immunogenicity score (TIS) reduction of more than 10 compared to the reference acidic α-glucosidase of SEQ ID NO: 2; (b) an immunogenicity hit count (IHC) reduction of more than 2 compared to the reference acidic α-glucosidase of SEQ ID NO: 2; (c) a total immunogenicity score (TIS) reduction of more than 10 compared to the reference acidic α-glucosidase of SEQ ID NO: 12; and / or (d) an immunogenicity hit count (IHC) reduction of more than 2 compared to the reference acidic α-glucosidase of SEQ ID NO: 12. 8. The engineered acidic α-glucosidase according to any one of claims 1 to 7, comprising the precursor peptide of the engineered acidic α-glucosidase. 9. The engineered acidic α-glucosidase of claim 8, wherein the precursor peptide of the engineered acidic α-glucosidase comprises a eukaryotic or synthetic signal peptide sequence. 10. The engineered acidic α-glucosidase of claim 9, wherein the signal peptide comprises a mouse or human signal peptide sequence. 11. The engineered acidic α-glucosidase of any one of claims 1 to 7, comprising the precursor peptide of the engineered acidic α-glucosidase. 12. The engineered acidic α-glucosidase of any one of claims 1 to 11, wherein the engineered...The acidic α-glucosidase is purified. 13. A pharmaceutical composition comprising the engineered acidic α-glucosidase according to any one of claims 1 to 12. 14. The pharmaceutical composition according to claim 13, further comprising a pharmaceutically acceptable carrier and / or excipient. 15. The pharmaceutical composition according to any one of claims 13 or 14, wherein the composition is suitable for parenteral injection or infusion into a human. 16. A recombinant polynucleotide comprising a polynucleotide sequence encoding the engineered acidic α-glucosidase according to any one of claims 1 to 11. 17. The recombinant polynucleotide of claim 16, comprising a reference polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, or 99% or higher sequence identity with a reference polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with a reference polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 81%, 82%, 83%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with a reference polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with a reference polynucleotide sequence having at least 70%, 75 ...6%, 97%, 98%, or 99% sequence identity with a reference polynucleotide sequence having at least 19. The recombinant polynucleotide of claim 16, comprising: a polynucleotide sequence comprising nucleotide residues 58 to 2832 of SEQ ID NOs with odd numbers from SEQ ID NO: 13 to 753; or a polynucleotide sequence comprising SEQ ID NOs with odd numbers from SEQ ID NO: 13 to 753. 20. The recombinant polynucleotide of claim 16, comprising: a polynucleotide sequence comprising nucleotide residues 58 to 2832 of SEQ ID NO: 13, 113, 125, 169, 249, 251, 393, 471, 487, or 505; or a polynucleotide sequence comprising SEQ ID NO: 13, 113, 125, 169, 249, 251, 393, 471, 487, or 505. 21. An expression vector comprising a recombinant polynucleotide according to any one of claims 16 to 20. 22. The expression vector of claim 21, wherein the recombinant polynucleotide is operatively linked to a control sequence.23. The expression vector of claim 22, wherein the control sequence comprises a promoter. 24. The expression vector of claim 23, wherein the promoter is a heterologous promoter. 25. A host cell comprising the expression vector of any one of claims 21 to 24. 26. The host cell of claim 25, wherein the host cell is a eukaryotic cell or a prokaryotic cell. 27. The host cell of claim 25, wherein the host cell is a mammalian cell. 28. The host cell of claim 27, wherein the mammalian cell is a human cell. 29. The host cell of claim 28, wherein the human cell is derived from a patient suffering from acid α-glucosidase activity deficiency. 30. A method for producing an engineered acid α-glucosidase variant, the method comprising culturing a host cell of any one of claims 25 to 29 under conditions that produce the acid α-glucosidase encoded by the recombinant polynucleotide. 31. The method of claim 30, further comprising the step of recovering the acid α-glucosidase. 32. The method of claim 30 or 31, further comprising the step of purifying the acidic α-glucosidase. 33. A method for treating and / or preventing symptoms of acidic α-glucosidase deficiency in a subject, the method comprising administering to a subject in need an effective amount of an engineered acidic α-glucosidase according to any one of claims 1 to 12 or a pharmaceutical composition according to any one of claims 13 to 15. 34. The method of claim 33, wherein the acidic α-glucosidase deficiency is Pompe disease. 35. The method of any one of claims 33 or 34, wherein the subject is an infant or child. 36. The method of any one of claims 33 or 34, wherein the subject is an adult or adolescent. 37. Use of the engineered acidic α-glucosidase according to any one of claims 1 to 12 for the treatment of acidic α-glucosidase deficiency. 38. Use of the engineered acidic α-glucosidase according to any one of claims 1 to 12 in the preparation of a medicament for treating a subject suffering from acidic α-glucosidase deficiency. 39. The use according to claim 37 or 38, wherein the acid α-glucosidase deficiency is Pompe disease. Claims 3 / 3 Page 4 CN 121511299 A Engineered acid α-glucosidase variant

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 511,347, filed June 30, 2023, which is incorporated herein by reference in its entirety.

[0003] Reference to sequence listings, tables, or computer programs

[0004] A sequence listing, filed concurrently with this document as filename CX7-223WO1_ST26.xml, created on June 27, 2024, and measuring 2,391,488 bytes, is part of the specification and is incorporated herein by reference. Technical Field

[0005] This disclosure relates to engineered acid α-glucosidase (GAA) polypeptides, compositions thereof, polynucleotides encoding engineered acid α-glucosidase polypeptides, and the use of engineered polypeptides for therapeutic and other purposes. Background Art

[0006] Pompe disease is an autosomal recessive lysosomal storage disease caused by mutations in the gene encoding acid α-glucosidase (GAA). This genetic defect results in a reduction or absence of acid α-glucosidase in body tissues. The resulting accumulation of glycogen in lysosomes causes lysosomal swelling and rupture, which can lead to cell damage, organelle dysfunction, and other cellular defects. Pompe disease has two main forms, including classic infantile and late-onset (childhood or adult) types, with some patients exhibiting an intermediate phenotype. The severity of the disease is related to the amount of enzyme activity present in the cells of the affected individual. The infantile form is the most severe and rapidly progressive, typically with acid α-glucosidase activity below 1%, leading to significant accumulation of glycogen in skeletal muscle, as well as in the heart and other tissues (see, for example, Hahn and Schänzer, Ann. Transl. Med., 2019, 7:283). In these patients, there is a multisystemic accumulation of lysosomal and nonlysosomal bound glycogen in the heart, skeletal muscle, and brain tissue (see, for example, Schoser, Ann. Transl. Med., 2019, 7:292). Patients exhibit elevated creatine kinase levels, hypertrophic cardiomyopathy, growth retardation, hypotonia, and axial muscle weakness. Without treatment, patients typically die within one year of birth from cardiopulmonary insufficiency. Survival beyond 18 months is extremely rare.

[0007] The infantile form differs from nonclassical or late-onset infantile Pompe disease, in which patients exhibit much milder myocardial hypertrophy. Patients with late-onset Pompe disease typically experience progressive limb-girdle myopathy and respiratory dysfunction. These patients exhibit marked but not unique muscle involvement. Patients eventually become wheelchair- and / or mechanically dependent. Respiratory failure is the leading cause of death in these patients. Some patients may synthesize nonfunctional acid α-glucosidase, but others do not produce any immune substances that cross-react with the native enzyme.

[0008] The human gene encoding acid α-glucosidase has been located on chromosomes 17q25.2–q25.3 and has been cloned and sequenced (see, for example, Peruzzo et al., Ann. Transl. Med., 2019, 7:278–287; and Martiniuk et al., DNA Cell. Biol., 1991,10:283-292). Although multiple mutations in this gene have been reported, the pathological mechanisms causing the various phenotypes observed in affected patients remain unclear. Despite the availability of enzyme replacement therapy (ERT) using recombinant human acid α-glucosidase, better treatment and management options for affected patients are still needed. Summary of the Invention

[0009] This disclosure provides an acid α-glucosidase polypeptide engineered to have improved properties, particularly compared to naturally occurring human acid α-glucosidase.

[0010] In some embodiments, this disclosure provides an engineered acidic α-glucosidase polypeptide or a bioactive fragment thereof comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with an even-numbered SEQ ID NO. in SEQ ID NO: 2, 12, 14, 754, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2 or relative to the reference sequence corresponding to SEQ ID NO: 12 or 2.

[0011] In some embodiments, the engineered acidic α-glucosidase or its bioactive fragment comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 12 or 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2 or relative to the reference sequence corresponding to SEQ ID NO: 12 or 2.

[0012] In some embodiments, the engineered acidic α-glucosidase polypeptide comprises a reference sequence of residues 20 to 944 of SEQ ID NO. corresponding to even-numbered residues in SEQ ID NO. 14 to 754, or having a content of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, or 91% of SEQ ID NO. corresponding to even-numbered residues in SEQ ID NO. 14 to 754.An amino acid sequence with 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher sequence identity, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2 or relative to the reference sequence corresponding to SEQ ID NO: 12 or 2.

[0013] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase includes at least substitutions at amino acid positions 24, 28, 29, 39, 50, 62, 78, 87, 135, 150, 266, 267, 305, 437, 486, 522, 569, 670, 692, 711, 736, 750, 812, 830, 842, 871, 883, 894, 913 or 932 or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or relative to the reference sequence corresponding to SEQ ID NO: 12.

[0014] In some embodiments, the amino acid sequence of the engineered acid α-glucosidase includes at least the substitutions for 24A / C / D / E / F / G / H / I / K / L / M / N / P / R / S / T / V / Y, 28A / C / D / E / F / G / H / K / L / P / Q / R / T / V / W, 29A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y, 39A / E / F / G / I / L / N / T, 50A / C / D / E / F / G / H / I / K / L / M / N / Q / R / S / Y. T / W / Y, 62A / D / E / F / G / H / I / K / M / N / P / Q / S / T / V / Y, 78A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / V / W / Y, 87A / D / G / H / I / K / L / MN / Q / R / S / T / V / W, 135A / C / D / E / F / G / H / I / K / L / N / P / R / Y, 150T, 266A / D / E / H / K / Q / T, 267H / L / R / T / V, 305V, 437A / H / S, 486A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 522A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y, 569A / C / D / E / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y, 670A / D / E / F / G / H / I / K / L / M / N / Q / R / S / V / Y, 692A / C / D / E / F / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 711A / C / D / E / F / G / I / K / L / M / N / Q / R / S / T / V / W / Y, 736F / L, 750A / E / K / L / Q / R, 812A / D / G / S, 830D / E / F / G / H / L / M / N / Q / S / T / V / W / Y, 842A / C / D / E / F / G / H / K / L / M / N / Q / R / T / W, 871A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 883A / F / Q, 894A / C / D / E / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 913E / F / H / I / K / M / N / Q / S / W or 932C / D / E / G / H / K / L / M / N / P / Q / R / S / T / W / Y, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or relative to the reference sequence corresponding to SEQ ID NO: 12. Instruction manual 2 / 115 pages 6 CN 121511299 A

[0015] In some embodiments, the amino acid sequence of the engineered acid α-glucosidase includes at least the substitutions 24A / C / D / F / G / H / I / K / M / N / P / S / T / V / Y, 28A / C / D / E / F / G / H / K / Q / T / V / W, 29A / C / D / E / F / G / H / I / K / M / N / P / R / W / Y, 39A / E / F / G / I / L / N / T, 50A / C / D / E / F / H / I / K / M / N / R / S / T / W / Y, 62D / H / I / K / M / N / P / Q / Y, 78A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 87A / G / H / I / K / L / MN / Q / R / S / T / V / W, 135C / D / E / F / G / H / I / K / L / N / R / Y, 266A / D / E / H / K / Q, 267H / L / T / V, 305V, 437A / H, 486C / D / F / G / H / I / K / L / M / N / Q / R / S / V / W / Y, 522A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y, 569A / C / D / E / G / K / M / N / P / R / W, 670A / D / G / H / K / M / Y, 692A / D / E / H / K / L / M / N / T / W, 711D / E / I / K / M / N / Q / S / T / V / Y, 736F / L, 750E / K / L / Q / R, 812A / D / G / S, 830D / E / F / G / H / L / M / N / S / T / W / Y, 842A / C / D / F / H / K / L / M / N / Q / R / T / W, 871A / C / D / F / H / I / M / N / Q / T / V / W / Y, 883A / F / Q, 894A / D / E / H / I / K / L / M / N / S / T / V / W / Y, 913F / I / K / M / N / S or 932C / D / E / G / H / K / L / M / N / P / Q / R / W / Y, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or relative to the reference sequence corresponding to SEQ ID NO: 12.

[0016] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase comprises a sequence including residues 20 to 944 of SEQ ID NO: 14 to 754 with even numbers, or a sequence including SEQ ID NO: 14 to 754 with even numbers.

[0017] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase comprises a sequence of residues 20 to 944 including SEQ ID NO: 14, 114, 126, 170, 250, 252, 394, 472, 488, or 506, or a sequence including SEQ ID NO: 14, 114, 126, 170, 250, 252, 394, 472, 488, or 506.

[0018] In some embodiments, the engineered acidic α-glucosidase exhibits at least one improved property compared to a reference acidic α-glucosidase. In some embodiments, compared with a reference acidic α-glucosidase having a sequence corresponding to residues 20 to 944 of SEQ ID NO: 2 or 12 or a sequence corresponding to SEQ ID NO: 2 or 12, the engineered acidic α-glucosidase exhibits at least one improved property selected from the following: i) enhanced catalytic activity; ii) improved tolerance to pH 7; iii) improved tolerance to pH 4.4; iv) improved stability in lysosomes; v) improved expression in cells; vi) improved uptake into cells; vii) improved enzymatic activity in cell lysates; viii) improved stability in plasma / serum; and ix) reduced immunogenicity; or a combination of any one of i), ii), iii), iv), v), vi), vii), viii), and ix).

[0019] In another aspect, this disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding the engineered acidic α-glucosidase disclosed herein.

[0020] In some embodiments, the recombinant polynucleotide comprises a reference polynucleotide sequence of nucleotide residues 58 to 2832 corresponding to odd-numbered SEQ ID NOs in SEQ ID NOs: 13 to 753, or a sequence of nucleotide residues 58 to 2832 corresponding to odd-numbered SEQ ID NOs in SEQ ID NOs: 13 to 753.The reference polynucleotide sequence of the odd-numbered SEQ ID NO. in 753 has a polynucleotide sequence with at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher sequence identity, wherein the polynucleotide encodes an acidic α-glucosidase.

[0021] In some embodiments, the recombinant polynucleotide encoding an engineered acidic α-glucosidase comprises a polynucleotide sequence codon-optimized for the expression of an engineered acidic α-glucosidase.

[0022] In some embodiments, the recombinant polynucleotide comprises: a polynucleotide sequence comprising nucleotide residues 58 to 2832 of the odd-numbered SEQ ID NO. in SEQ ID NO: 13 to 753; or a polynucleotide sequence comprising the odd-numbered SEQ ID NO. in SEQ ID NO: 13 to 753.

[0023] In another aspect, this disclosure provides an expression vector comprising a recombinant polynucleotide encoding the engineered acid α-glucosidase described herein. In some embodiments, the expression vector comprises a control sequence operatively linked to the recombinant polynucleotide encoding the engineered acid α-glucosidase. In some embodiments, the control sequence is a promoter, such as a heteropromoter.

[0024] In another aspect, this disclosure provides a host cell comprising an expression vector containing the recombinant polynucleotide described herein. In some embodiments, the host cell is a eukaryotic or prokaryotic cell. In some embodiments, the host cell is a mammalian cell, particularly a human cell. In some embodiments, the human cell is derived from a patient with an acid α-glucosidase activity deficiency, such as a patient with Pompe disease.

[0025] In another aspect, the host cell is used to produce the engineered acid α-glucosidase disclosed herein. In some embodiments, a method for producing an engineered acidic α-glucosidase includes culturing a host cell containing an expression vector under suitable conditions for producing an engineered acidic α-glucosidase.

[0026] In another aspect, this disclosure provides a pharmaceutical composition comprising an engineered acidic α-glucosidase or a recombinant polynucleotide encoding an engineered acidic α-glucosidase, comprising an expression vector containing the recombinant polynucleotide. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0027] In another aspect, an engineered acidic α-glucosidase or a recombinant polynucleotide encoding an engineered acidic α-glucosidase...Nucleotides are used to treat subjects suffering from acid alpha-glucosidase activity deficiency. In some embodiments, a method for treating and / or preventing symptoms of acid alpha-glucosidase deficiency in a subject includes administering to a subject in need an effective amount of an engineered acid alpha-glucosidase or a recombinant polynucleotide encoding an engineered acid alpha-glucosidase disclosed herein.

[0028] In some embodiments, a pharmaceutical composition comprising an engineered acid alpha-glucosidase or a recombinant polynucleotide encoding an engineered acid alpha-glucosidase is administered to a subject.

[0029] In some embodiments, the subject receiving treatment suffers from Pompe disease. In some embodiments, the subject is an infant or a child. In some embodiments, the subject is an adult or an adolescent.

[0030] In another aspect, this disclosure provides the use of an engineered acid alpha-glucosidase or a recombinant polynucleotide encoding an engineered acid alpha-glucosidase, or a pharmaceutical composition thereof, for the treatment of acid alpha-glucosidase activity deficiency. In some embodiments, acid alpha-glucosidase deficiency is Pompe disease.

[0031] Figure 1 provides a graph showing the duration of stability of the six GAA variants in neutral pH cell culture medium, as described in Example 6.

[0032] Figure 2 provides a graph showing the melting temperatures of the six GAA variants at lysosomal (4.4) and weakly alkaline (7.4) pH, as described in Example 6.

[0033] Figure 3 provides a graph showing the duration of stability of the six GAA variants upon stimulation by treatment with plasma, as described in Example 6.

[0034] Figure 4 provides a graph showing the 4-MU-GLU hydrolytic activity in lysates of Pompeii patient-derived fibroblasts cultured with the seven purified GAA variants for 1 hour (small figure 4A), 4 hours (small figure 4B), 24 hours (small figure 4C), or 96 hours (small figure 4D), after washing off the GAA material and incubating at 37°C until 96 hours post-treatment, as described in Example 6. Values ​​are expressed as RFU activity.

[0035] Figure 5 provides a graph showing the 4-MU-GLU hydrolytic activity in lysates of cultured C2C12 GAA knockout myoblasts treated with seven purified GAA variants for 1 hour (inset 5A), 4 hours (inset 5B), 24 hours (inset 5C), or 96 hours (inset 5D), after washing off the GAA material and incubating at 37°C for 96 hr post-treatment, as described in Example 6. Values ​​are expressed as RFU activity.

[0036] Figure 6 provides a graph showing the 4-MU-GLU hydrolytic activity in the supernatant of cultures of C2C12 GAA knockout myoblasts transfected with plasmid DNA of six GAA variants.

[0037] Figure 7 provides a graph illustrating GAA activity in normalized lysates of C2C12 GAA knockout myoblasts transfected with plasmid DNA of six GAA variants (4-MU-GLU hydrolysis shown in inset 7A and glycogen hydrolysis shown in inset 7B).

[0038] Figure 8 shows, in inset 8A, the sum of all high-quality GAA-derived peptides observed by mass spectrometry from each GAA variant in an in vitro MHC II-related peptide proteomics assay (MAPP assay); and in inset 8B, a plot of peptide frequencies across donors for each GAA variant relative to the GAA sequence regions from which the peptides are processed. In the MAPP assay, PBMCs from healthy donors were differentiated into dendritic cells (antigen-presenting cells) and incubated in the presence of GAA variants (antigens). HLA-binding peptides were then eluted from HLA-DR molecules and identified by mass spectrometry, providing information about peptide processing and presentation in antigen-presenting cells. These results indicate that, compared to WT GAA (SEQ ID NO: 2), the GAA variants of SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 14 exhibit significantly reduced processing and peptide presentation frequency. Detailed Description

[0039] This disclosure provides engineered acidic α-glucosidase (GAA) peptides and compositions thereof. In some embodiments, the engineered acidic α-glucosidase peptides are engineered to exhibit improved properties, including enhanced catalytic activity and enhanced acid stability, while reducing sensitivity to proteolysis. This disclosure also provides methods for using engineered acidic α-glucosidase peptides (including compositions thereof) for therapeutic and other purposes.

[0040] Abbreviations and Definitions

[0041] Unless otherwise expressly defined, the technical and scientific terms used in this disclosure will have the meanings commonly understood by one of ordinary skill in the art.

[0042] It should be understood that the invention described herein is not limited to the specific methods, schemes, and reagents described, as these may vary depending on the context in which they are used by those skilled in the art. Therefore, the following terms, defined below, are described more fully by reference to the entire application.

[0043] Furthermore, the section headings provided herein should not be construed as limiting on various aspects or embodiments of the invention, which can be obtained by referring to the entire application.

[0044] As used herein, unless the context clearly indicates otherwise, the singular “a,” “an,” and “the” include plural indicators.

[0045] As used herein, the term “comprising” and its cognates are used in their inclusive sense (i.e., equivalent to the term “comprising” and its corresponding cognates).

[0046] It should also be understood that when the term “comprising” and its cognates are used in the description of embodiments, the term “main” may also be used.The embodiments are described as “to consist of” or “to be composed of”.

[0047] Furthermore, numerical ranges include numbers that define a range. Therefore, each numerical range disclosed herein is intended to cover each narrower numerical range falling within such a wider numerical range, as all such narrower numerical ranges are explicitly written herein. Additionally, each maximum (or minimum) numerical limit disclosed herein is intended to include each lower (or higher) numerical limit, as such lower (or higher) numerical limits are explicitly written herein.

[0048] “About” means an acceptable error for a particular value. In some cases, “about” means within 0.05%, 0.5%, 1.0%, or 2.0% of a given value range. In some cases, “about” means within 1, 2, 3, or 4 standard deviations of a given value. In some cases, “about” covers 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8% of a given value. Values ​​within 8.5%, 9%, 9.5%, or 10%.

[0049] The “EC” number refers to the nomenclature of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification system is a digital classification system for enzymes based on enzyme-catalyzed chemical reactions.

[0050] “ATCC” refers to the American Type Culture Collection, whose biological resource bank contains genes and strains.

[0051] “NCBI” refers to the National Center for Bioinformatics and its sequence database.

[0052] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein to refer to polymers of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation).

[0053] Amino acids are represented in this paper using their commonly known three-letter symbols or single-letter symbols recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature. The abbreviations for amino acids used in genetic coding are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glycine (Gly or G), glutamine (Gln or Q), histidine (His or H), and isoleucine (Ile or I). Leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). When using three-letter abbreviations, unless specifically preceded by "L" or "D" or clearly indicated from the context of the abbreviation, the meaning should be understood.Otherwise, the amino acid may be in the L- or D-configuration with respect to the α-carbon (Cα). For example, “Ala” represents alanine without specifying the α-carbon configuration, while “D-Ala” and “L-Ala” represent D-alanine and L-alanine, respectively. When using single-letter abbreviations, uppercase letters indicate amino acids in the L-configuration with respect to the α-carbon, while lowercase letters indicate amino acids in the D-configuration with respect to the α-carbon. For example, “A” represents L-alanine, and “a” represents D-alanine. When the polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or a combination thereof), the sequence is presented in the amino (N) to carboxyl (C) orientation according to common convention.

[0054] “fusion protein” and “chimeric protein” and “chimera” refer to hybrid proteins generated by linking two or more polynucleotides that originally encode a single protein. In some embodiments, fusion proteins are generated by recombinant techniques (e.g., molecular biology techniques known in the art).

[0055] “Acid alpha-glucosidase”, “acid alpha-glucosidase”, “acid alpha-glucosidase polypeptide”, “lysosomal alpha-glucosidase” and “GAA” refer to enzymes within the family of enzymes that break down glycogen present in lysosomes (EC 3.2.1.20). This enzyme is sometimes also referred to as “alpha-1,4-glucosidase”, “acid maltase”, “glucose invertase”, “glucosidase”, “lysosomal alpha-glucosidase”, “maltase”, or “maltase-glucosidase”. One reaction catalyzed by this enzyme is the hydrolysis of terminally non-reduced (1 to 4) linked α-D-glucose residues, releasing α-D-glucose. As used herein, the term “rhGAA” refers to recombinant human acid α-glucosidase.

[0056] “Pompe disease” refers to type II glycogen storage disease, which is usually an autosomal recessive genetic disorder that causes metabolic disturbances, characterized by lysosomal accumulation of glycogen in skeletal muscle and other tissues. It is characterized based on age of onset, organ involvement, severity, and rate of progression. A more severe type is infancy-onset Pompe disease (IOPD), which occurs in infants. Another type, called “late-onset Pompe disease” (LOPD), occurs in individuals who develop the disease before 12 months of age but without the cardiomyopathy associated with IOPD, and in all individuals who develop the disease after 12 months of age. Synonyms for Pompe disease include “acid α-glucosidase deficiency”, “acid maltose deficiency”, etc.Enzyme deficiency, GAA deficiency, type II glycogen storage disease, GSD II, GSD2, and type II glycogen disease.

[0057] The terms “polynucleotide,” “nucleic acid,” or “oligonucleotide” are used herein to refer to polymers containing at least two nucleotides, wherein the nucleotides are deoxyribonucleotides or ribonucleotides or a mixture of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations for genetically encoded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless otherwise expressly specified, the abbreviation for nucleoside may be ribonucleoside or 2'-deoxyribonucleoside. Nucleosides may be specified alone or in combination as ribonucleoside or 2'-deoxyribonucleoside. When polynucleotides, When nucleic acid or oligonucleotide sequences are presented as single-letter abbreviation strings, the sequences are conventionally presented in a 5' to 3' orientation and do not indicate phosphate. The term "DNA" refers to deoxyribonucleic acid. The term "RNA" refers to ribonucleic acid. Polynucleotides or nucleic acids can be single-stranded or double-stranded, or may include both single-stranded and double-stranded regions.

[0058] When used to refer to cells, polynucleotides, or polypeptides, "engineered," "recombinant," "non-naturally occurring," and "variant" refer to a material or a material corresponding to a natural or native form that has been modified in a manner that would not exist in nature in any other form or in the same way, but is produced or derived from synthetic materials and / or manipulated using recombinant techniques.

[0059] "Wild-type" and "naturally occurring" refer to forms that exist 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. In some embodiments, "wild-type" and "naturally occurring" refer to forms found in nature that have normal function and / or activity.

[0060] “Coding sequence” refers to a portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.

[0061] “Sequence identity percentage (%)” is used herein to refer to comparisons between polynucleotides and peptides and is determined by comparing two best-aligned sequences in a comparison window, wherein the portion of the polynucleotide or peptide sequence in the comparison window may include additions or deletions (i.e., vacancies) relative to a reference sequence used for the best-alignment of the two sequences. The percentage is calculated by determining the number of positions in the two sequences where the same nucleic acid base or amino acid residue appears to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Alternatively, the percentage can be calculated by determining the number of positions in the two sequences where the same nucleic acid base or amino acid residue appears to be present.The number of positions of bases or amino acid residues, or the number of positions where nucleic acid bases or amino acid residues are aligned with vacancies, is used to generate the number of matching positions. This number of matching positions is then divided by the total number of positions in the comparison window, and the result is multiplied by 100 to obtain the sequence identity percentage. Those skilled in the art will understand that many established algorithms are available for aligning two sequences. The best alignment of sequences for comparison can be performed, for example, by: Smith and Waterman’s local homology algorithm (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), Needleman and Wunsch’s homology alignment algorithm (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), Pearson and Lipman’s similarity search method (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or visual inspection, as known in the art. Examples of algorithms suitable for determining sequence identity and sequence similarity percentages include, but are not limited to, the BLAST and BLAST 2.0 algorithms, described by Altschul et al. (see Altschul et al., J. Mol. Biol., 1990, 215:403–410; and Altschul et al., Nucleic Acids Res., 1997, 25(17):3389–3402, respectively). Software for performing BLAST analysis is publicly available from the website of the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence. When compared with words of the same length in the database sequence, the short word matches or satisfies a positive threshold score T. T is called the neighborhood character score threshold (see Altschul et al., ibid.). These initial neighborhood character hits act as seeds to initiate a search to find longer HSPs containing those initial neighborhood character hits. Then the word hit extends in both directions along each sequence until the cumulative alignment score can be increased as per the specification 7 / 115 page 11 CN 121511299 A. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a mismatch; always <0). For amino acid sequences, a score matrix is ​​used to calculate the cumulative score. When a word hit occurs...The extension of word hits in each direction will stop under the following conditions: the cumulative alignment score decreases by an amount X from its maximum realized value; the cumulative score becomes zero or lower due to the accumulation of one or more negatively scored residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expected value (E) of 10, M = 5, N = -4, and comparisons of two strands as default values. For amino acid sequences, the BLASTP program defaults to a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary determinations of sequence alignment and sequence identity percentage can be performed using the BESTFIT or GAP procedures in the GCG Wisconsin software package (Accelrys, Madison WI, Inc., Accelrys, WI).

[0062] A “reference sequence” refers to a defined sequence used as the basis for sequence comparison. A reference sequence can be a subset of a larger sequence, such as a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is at least 20 nucleotides or amino acid residues long, at least 25 residues long, at least 50 residues long, at least 100 residues long, or the full length of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) contain sequences similar to those between the two sequences (i.e., a portion of the complete sequence) and (2) may further contain sequences dissimilar to those between the two sequences, sequence comparisons between these two (or more) polynucleotides or polypeptides are typically performed by comparing the sequences of the two polynucleotides or polypeptides on a “comparison window” to identify and compare local regions with sequence similarity. In some embodiments, a “reference sequence” may be based on a primary amino acid sequence, wherein the reference sequence is a sequence that may have one or more alterations to the primary sequence.

[0063] A “comparison window” refers to a conceptual segment of consecutive nucleotide positions or amino acid residues, wherein the sequence can be compared with a reference sequence. In some embodiments, the comparison window is at least 15 to 20 consecutive nucleotides or amino acids, and wherein a portion of the sequence in the comparison window may include 20% or less of additions or deletions (i.e., vacancies) compared to the reference sequence (excluding additions or deletions) to allow for optimal alignment of the two sequences. In some embodiments, the comparison window may be greater than 15 to 20 consecutive residues, and optionally includes windows of 30, 40, 50, 100 or longer.

[0064] When used in the context of numbering a given amino acid or polynucleotide sequence, “corresponding to”, “reference”, etc."Reference" or "relative to" refers to the numbering of residues in a specified reference sequence when comparing a given amino acid or polynucleotide sequence to a reference sequence. In other words, the residue numbering or position of a given polymer is specified relative to a reference sequence rather than based on the actual numerical position of residues within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence (such as the sequence of an engineered acidic α-glucosidase) can be aligned to a reference sequence by introducing vacancies to optimize residue matching between two sequences. In these cases, the numbering of residues in the given amino acid or polynucleotide sequence is relative to the aligned reference sequence, despite the presence of vacancies.

[0065] "Mutation" refers to an alteration of a nucleic acid sequence. In some embodiments, a mutation causes a change in the encoded polypeptide sequence (i.e., compared to the original sequence without mutation). In some embodiments, a mutation includes substitution, resulting in a different amino acid. In some alternative embodiments, a mutation includes addition, resulting in the addition (e.g., insertion) of an amino acid into the original polypeptide sequence. In some other embodiments, a mutation includes deletion, resulting in the deletion of an amino acid from the original polypeptide sequence. Any number of mutations may be present in a given sequence.

[0066] "Amino acid difference" or "residue difference" refers to the difference between an amino acid residue at a certain position in a polypeptide sequence and the corresponding amino acid residue at a position in a reference sequence. The position of the amino acid difference is usually referred to as "Xn" in this document, where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "residue difference at position X24 compared to SEQ ID NO: 12" refers to the difference in amino acid residues at position X24 of the polypeptide compared to position X24 of SEQ ID NO: 12. Therefore, if the reference polypeptide of SEQ ID NO: 12 has tryptophan at position X24, then "residue difference at position X24 compared to SEQ ID NO: 2" refers to the difference in amino acid residues at position X24 of the polypeptide, excluding those at position X24 of SEQ ID NO: 12. The amino acid substitution at position 12, corresponding to position 24, for any residue other than tryptophan. In most cases herein, a specific amino acid residue difference at a position is indicated as “XnY”, where “Xn” specifies the corresponding position as described above, and “Y” is a single-letter identifier of the amino acid found in the engineered peptide (i.e., a residue different from the reference peptide). In some cases (e.g., as shown in Table 3-1), this disclosure also provides for a specific amino acid difference represented by the conventional symbol “AnB”, where A is a single-letter identifier of a residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is a single-letter identifier of a residue substitution in the sequence of the engineered peptide. In some embodiments, amino acid differences (e.g., substitutions) are represented by the abbreviation “nB”.The reference sequence is indicated without including the identifier of the residue in the reference sequence. In some embodiments, the phrase “amino acid residue nB” indicates the presence of an amino acid residue in the engineered polypeptide, which may or may not be a substitution in the context of the reference sequence.

[0067] In some cases, the polypeptide of this disclosure may include one or more amino acid residue differences relative to the reference sequence, indicated by a list of designated positions where the residue difference occurs relative to the reference sequence. In some embodiments, when more than one amino acid may be used at a particular residue position of the polypeptide, the various amino acid residues that may be used are separated by “ / ” (e.g., X24A / X24C or X24A / C or 24A / C). In some embodiments, the amino acid residue is selected from the various alternative amino acid residues listed at the residue position. In some embodiments, the polypeptide variant contains more than one substitution. For readability, these substitutions are separated by slashes (e.g., L24W / L28S or 24W / 28S) or by semicolons, as described below. As previously mentioned, in some cases, the position number in this application is not preceded by an “X”.

[0068] “Amino acid substitution set” and “substitution set” refer to a group of amino acid substitutions within the polypeptide sequence. In some embodiments, the substitution set comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. In some embodiments, the substitution set refers to the set of amino acid substitutions in any of the acidic α-glucosidase polypeptides listed in any of the tables in the examples. In these substitution sets, each substitution is separated by a semicolon (e.g., L24W; L28S) or a forward slash (" / "; e.g., L24W / L28S or 24W / 28S). In some embodiments, "substitution" includes the deletion of an amino acid and may be indicated by a "-" symbol.

[0069] "Conservative amino acid substitution" refers to the substitution of a residue with a different residue having a similar side chain, and therefore generally involves the substitution of an amino acid in a polypeptide with an amino acid within the same or similarly defined amino acid class. For example, but not limited to, an amino acid with an aliphatic side chain may be replaced by another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain may be replaced by another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain may be replaced by another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain may be replaced by another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain may be replaced by another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid may be replaced by another hydrophobic or hydrophilic amino acid, respectively.

[0070] "Non-conservative substitution" refers to replacing an amino acid in a polypeptide with an amino acid whose side chain characteristics are significantly different. Non-conservative substitution can be used between rather than within defined groups of amino acids and affects (a) the structure of the peptide backbone in the substituted region (e.g., replacing glycine with proline), (b) charge or hydrophobicity, or (c) the majority of the side chain. For example, but not limited to, exemplary non-conservative substitutions can be the substitution of acidic amino acids with basic or aliphatic amino acids; the substitution of aromatic amino acids with small amino acids; and the substitution of hydrophilic amino acids with hydrophobic amino acids.

[0071] "Deficiency" refers to modifying a polypeptide by removing one or more amino acids from a reference polypeptide. Deficiency can include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, representing up to 10% or up to 20% of the total number of amino acids in the reference polypeptide, while maintaining activity and / or maintaining the improved properties of the engineered polypeptide. Deletions can target internal portions and / or terminal portions of a polypeptide. In various embodiments, deletions can comprise continuous segments or can be discontinuous.

[0072] An "insertion" refers to modifying a polypeptide by adding one or more amino acids from a reference polypeptide. An insertion can be located within an internal portion of the polypeptide or at the carboxyl or amino terminus. Insertions as used herein include fusion proteins as known in the art. An insertion can be a continuous segment of amino acids or can be separated by one or more amino acids from naturally occurring polypeptides.

[0073] The terms "functional fragment" or "bioactive fragment," used interchangeably herein, refer to a polypeptide having amino-terminal and / or carboxyl-terminal deletions and / or internal deletions, but wherein the remaining amino acid sequence is identical to the corresponding amino acid sequence in the compared sequence (e.g., the full-length engineered acid α-glucosidase of the present invention) and retains virtually all the activity of the full-length polypeptide.

[0074] An "isolated polypeptide" refers to a polypeptide that has been substantially isolated from other naturally occurring contaminants (e.g., proteins, lipids, and polynucleotides). This term encompasses polypeptides that have been removed or purified from their natural environment or expression system (e.g., host cells or in vitro synthesis). Engineered acidic α-glucosidase polypeptides can be present in cells, in cell culture media, or prepared in various forms, such as lysates or isolated formulations. Thus, in some embodiments, engineered acidic α-glucosidase polypeptides can be isolated polypeptides.

[0075] "Substantially pure polypeptide" means a composition in which the polypeptide species is the dominant species present (i.e., its abundance, by molar or weight, is higher than that of any other individual macromolecular species in the composition), and when the target species is present by molar or weight...A composition is generally referred to as substantially purified when it comprises at least about 50% by weight of macromolecular species. Typically, a substantially pure polypeptide composition contains about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more by molar or weight percentage of all macromolecular species present in the composition. In some embodiments, the target species is purified to substantially homogeneity (i.e., contaminant species are not detectable in the composition by conventional detection methods), wherein the composition consists substantially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ionic species are not considered macromolecular species. In some embodiments, the isolated polypeptide is a substantially pure polypeptide composition.

[0076] "Improved enzyme properties" means that the engineered acid α-glucosidase polypeptide exhibits improvements in any enzyme properties compared to a reference acid α-glucosidase polypeptide, which may be a wild-type acid α-glucosidase polypeptide or another engineered acid α-glucosidase polypeptide. Improved properties include, but are not limited to, the following characteristics: increased protein expression, increased thermal activity, increased thermal stability, increased pH activity, increased stability, increased enzymatic activity, increased specific activity, enhanced resistance to substrate or end-product inhibition, improved chemical stability, improved solvent stability, improved tolerance to acidic, neutral, or alkaline pH, improved tolerance to proteolytic activity (i.e., reduced sensitivity to proteolysis), reduced aggregation, increased solubility, reduced immunogenicity, improved post-translational modifications (e.g., glycosylation), altered temperature profile, and improved lysosomal stability.

[0077] "Enhanced enzymatic activity" or "enhanced catalytic activity" refers to the improved properties of an engineered acidic α-glucosidase polypeptide, which can be represented by an increase in specific activity (e.g., product / time / weight protein produced) or a percentage conversion of substrate to product (e.g., percentage conversion of an initial amount of substrate to product when using a specific amount of acidic α-glucosidase over a specific time period). Exemplary methods for determining enzyme activity are provided in the examples. Any property related to enzyme activity can be affected, including classic enzyme properties such as Km, Vmax, or kcat, and changes in these properties may lead to an increase in enzyme activity. The increase in enzyme activity compared to naturally occurring acidic α-glucosidase or another engineered acidic α-glucosidase derived from an acidic α-glucosidase polypeptide can be approximately 1.1 times the enzyme activity of the corresponding wild-type enzyme, up to 2, 5, 10, 20, 25, 50, 75, 100, 150, 200, or more times.

[0078] In some embodiments, acidic α-glucosidase activity can be achieved by any suitable method known in the art.Measurements (e.g., standard assays, such as monitoring changes in the spectrophotometric properties of reactants or products). In some embodiments, the amount of product produced after hydrolysis of the 4-methylumbelliferyl-α-D-glucopyranoside (4-MUGlu) molecule can be measured by monitoring fluorescence (excitation 355 nm, emission 460 nm), as provided in the examples. Enzyme activity comparisons are performed using defined enzyme preparations, defined assays under set conditions, and one or more defined substrates, as described in detail herein. Typically, when comparing lysates, the number of cells and the amount of protein measured are determined, and the same expression system and the same host cells are used to minimize variability in the amount of enzyme produced by the host cells and present in the lysates.

[0079] "Increased tolerance to acidic pH" means that the engineered acidic α-glucopyranoside according to the invention will have improved stability (e.g., maintaining higher activity at about pH 4-4.8 after exposure to acidic pH for a specific period of time (e.g., 1 hour, up to 24 hours)) compared to a reference acidic α-glucopyranoside or another enzyme.

[0080] "Improved tolerance to neutral pH" means that the engineered acid α-glucosidase according to the invention will have improved stability (maintaining higher activity at approximately pH 7 after exposure to neutral pH for a specific period of time (e.g., 1 hour and up to 24 hours)) compared to a reference acid α-glucosidase or another enzyme.

[0081] "Improved cellular uptake" means that the engineered acid α-glucosidase provided herein exhibits improved endocytosis into cells compared to a reference acid α-glucosidase (including wild-type acid α-glucosidase) or another enzyme. In some embodiments, the cells are cultured Pompeii patient-derived cells (which maintain higher intracellular activity after incubation with cultured cells for a specific period of time compared to a reference acid α-glucosidase or another enzyme). In some other embodiments, the engineered acid α-glucosidases provided herein exhibit higher intracellular activity with cultured cells over a specific time period compared to a reference acid α-glucosidase (including wild-type acid α-glucosidase) or another enzyme. In some alternative embodiments, the time period is about 4 hours, while in some other embodiments, the time period is less than 4 hours (e.g., 1, 2, or 3 hours), and in some alternative embodiments, the time period is greater than 4 hours (e.g., 5, 6, 7, 8 hours, or more).

[0082] "Reduced immunogenicity" and "weakened immunogenicity" refer to the engineered acid α-glucosidases provided herein inducing or expected to reduce the immune response compared to wild-type or another reference acid α-glucosidase.

[0083] As used herein, "physiological pH" refers to the pH range typically found in the blood of a subject (e.g., a human).

[0084] "Neutral pH" (e.g., used to refer to improved stability to alkaline pH conditions or enhanced tolerance to alkaline pH) means a pH of approximately 7.

[0085] "Alkaline pH" (e.g., used to refer to improved stability to alkaline pH conditions or enhanced tolerance to alkaline pH) means a pH > 7, for example, in the pH range of > 7 to 11.

[0086] "Acidic pH" (e.g., used to refer to improved stability to acidic pH conditions or enhanced tolerance to acidic pH) means a pH < 7, for example, in the pH range of approximately 1.5 to 4.8.

[0087] "Conversion" refers to the enzymatic conversion (or bioconversion) of a substrate into the corresponding product. "Conversion percentage" refers to the percentage of substrate converted to product under specific conditions over a given time period. Therefore, the "enzymatic activity" or "activity" of an acidic α-glucosidase polypeptide can be expressed as the "conversion percentage" of substrate converted to product over a given time period.

[0088] "Suitable reaction conditions" refers to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffer, cosolvent, etc.) under which the acidic α-glucosidase polypeptide of this application can convert the substrate into the desired product compound. Exemplary "suitable reaction conditions" are provided in this application and illustrated by example. "Loading," such as "compound loading" or "enzyme loading," refers to the concentration or amount of a component in the reaction mixture at the start of the reaction. In the context of an enzymatic conversion reaction process, "substrate" refers to a compound or molecule acted upon by the acidic α-glucosidase polypeptide. In the context of an enzymatic conversion reaction process, "product" refers to a compound or molecule produced after the acidic α-glucosidase polypeptide acts on the substrate.

[0089] "Codon-optimized" means changing the codons of a polynucleotide encoding a protein to codons preferred by a particular organism, so that the encoded protein is expressed more effectively in the organism of interest. Although the genetic code is degenerate, as most amino acids are represented by several codons (called "synonyms" or "synonyms"), it is well known that codon usage in a particular organism is non-random and biased towards specific codon triplets. This codon usage bias can be even greater for a given gene, genes with a common function or ancestral origin, highly expressed proteins relative to low copy number proteins, and protein-coding regions of an organism's genome. In some embodiments, the polynucleotide encoding acid α-glucosidase can be codon-optimized to obtain optimal yield from a selected host organism for expression.

[0090] As used herein, "control sequence" refers to all components necessary or advantageous for the expression of the polynucleotides and / or polypeptides of this application. Each control sequence may be native or exogenous for the nucleic acid sequence encoding the polypeptide. Such control sequencesThe sequence includes, but is not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoter sequences, signal peptide sequences, initiation sequences, and transcription terminators. In some embodiments, the control sequence includes at least a promoter, as well as transcription and translation stop signals.

[0091] Herein, “operably linked” or “operably linked” is defined as a configuration in which the control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to the polynucleotide of interest, such that the control sequence directs or regulates the expression of the polynucleotide and / or, where appropriate, the encoded polypeptide of interest.

[0092] A “promoter sequence” refers to a nucleic acid sequence recognized by the host cell for the expression of the polynucleotide of interest (such as a coding sequence). The promoter sequence contains a transcription control sequence that mediates the expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutant, truncated, and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0093] A “vector” refers to a polynucleotide construct used to introduce a polynucleotide sequence into a cell. In some embodiments, the vector is an "expression vector" operatively linked to a suitable control sequence capable of enabling the expression of a polynucleotide of interest and / or a polypeptide encoded in the polynucleotide in a suitable host. In some embodiments, the expression vector has a promoter sequence operatively linked to a polynucleotide sequence (e.g., a transgene) to drive expression in a host cell, and in some embodiments, the expression vector also includes a transcription terminator sequence.

[0094] "Expression" encompasses any step involving polypeptide production, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also covers the secretion of polypeptides from cells.

[0095] "Cultivation" refers to the growth of a cell population under any suitable conditions (e.g., using liquid, gel, or solid culture media).

[0096] "Production" refers to the production or expression of proteins and / or other compounds by cells. The term is intended to cover any step involving polypeptide production, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also covers the secretion of polypeptides from cells.

[0097] "Heterologous" or "recombinant" refers to a relationship between two or more nucleic acid or protein sequences (e.g., promoter sequences, signal peptides, terminator sequences, etc.) that originate from different sources and are not related in nature.

[0098] "Host cell" and "host strain" refer to a host suitable for expressing the expression vector containing a polynucleotide provided herein (e.g., a polynucleotide encoding an acid α-glucosidase variant). In some embodiments, the host cell is the one described in the specification, page 12 / 115, 16 CN 121511299 A.Transformation or transfection of prokaryotic or eukaryotic cells with vectors constructed using recombinant DNA techniques known in the art.

[0099] "Therapeutic" means a compound having a beneficial or desirable medical effect when administered to a subject exhibiting pathological signs or symptoms.

[0100] "Gene therapy vector" means a medium or carrier suitable for delivering polynucleotides to cells to exert a therapeutic effect. In some embodiments, the vector encapsulates a gene (e.g., a therapeutic gene) or a polynucleotide sequence for delivery to cells or tissues, including but not limited to adenovirus (AV), adeno-associated virus (AAV), lentivirus (LV), and nonviral vectors such as liposomes. The present invention is not intended to be limited to any particular gene therapy vector, as any medium suitable for a particular situation may be used. Gene therapy vectors may be designed to deliver genes to a particular species or host, or may have broader applicability.

[0101] "Gene therapy" means the use of a gene therapy vector to deliver a gene, polydeoxynucleotide, or polynucleotide sequence to cells or tissues to modify those cells or tissues, thereby treating or preventing disease. Gene therapy may include replacing a mutated gene causing a disease with a healthy copy of the gene or a functional variant of the gene, or inactivating or “knocking out” a mutated gene that causes a disease. In some embodiments, gene therapy is used to treat a patient’s disease.

[0102] “mRNA therapy” refers to the delivery of an mRNA polynucleotide sequence to cells or tissues to modify those cells or tissues, thereby treating or preventing a disease. In some embodiments, the mRNA polynucleotide sequence for delivery to cells or tissues is formulated, for example, but not limited to, liposomes. In some embodiments, mRNA therapy is used to treat a patient’s disease.

[0103] “Cell therapy” refers to the delivery of exogenously modified live cells into a patient to provide a missing gene, thereby treating or preventing a disease. The modified cells are then reintroduced into the body.

[0104] “Composition” and “formulation” encompass products comprising at least one engineered acidic α-glucosidase of the present disclosure, intended for any suitable use (e.g., pharmaceutical composition, dietary / nutritional supplement, tonic, etc.).

[0105] A “pharmaceutical composition” means a pharmaceutical composition suitable for mammalian subjects (e.g., humans) that comprises a pharmaceutically effective amount of an engineered acidic α-glucosidase polypeptide covered by the present invention and an acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises a recombinant polynucleotide encoding an engineered acidic α-glucosidase, for example, in the form of a gene therapy vector.

[0106] “Pharmaceutical acceptable” means a substance that can be administered to a subject without causing any undesirable biological effects or interacting harmfully with any of the components contained therein and has the desired biological activity.

[0107] When used in a pharmaceutical composition, “carrier” means any of the standard pharmaceutical carriers, buffers, and excipients, such as stabilizers, preservatives, and adjuvants.

[0108] “Excipient” means any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient other than the active pharmaceutical ingredient (API; for example, an engineered acid α-glucosidase polypeptide or a recombinant polynucleotide encoding acid α-glucosidase). Excipients are typically included for formulation and / or administration purposes.

[0109] “Administration” and “administering” of a composition refer to providing the composition of the present invention to a subject (e.g., a person affected by Pompe disease).

[0110] “Effective amount” means an amount sufficient to produce the desired result. An effective amount can be determined by those skilled in the art using conventional experiments.

[0111] When used to refer to symptoms of a disease / symptom, “therapeutic effective amount” refers to the amount and / or concentration of a compound (e.g., an engineered acid α-glucosidase polypeptide or a recombinant polynucleotide encoding an engineered acid α-glucosidase) that improves, alleviates, or eliminates one or more symptoms of the disease / symptom, or prevents or delays the onset of symptoms. When used to refer to a disease / symptom, “therapeutic effective amount” refers to the amount and / or concentration of a composition (e.g., an engineered GAA polypeptide or a recombinant polynucleotide encoding an engineered acid α-glucosidase) that improves, alleviates, or eliminates the disease / symptom. In some embodiments, the term is used to refer to the amount of a composition that elicits a biological (e.g., medical) response in a tissue, system, or animal subject as desired by a researcher, physician, veterinarian, or other clinician.

[0112] As used herein, “treating” or “treatment” of a disease, condition, or syndrome includes (i) preventing the occurrence of a disease, condition, or syndrome in a subject, even if clinical symptoms of the disease, condition, or syndrome do not develop in animals that may be exposed to or susceptible to the disease, condition, or syndrome but have not yet experienced or exhibited symptoms of the disease, condition, or syndrome; (ii) suppressing the disease, condition, or syndrome, i.e., preventing its development; and (iii) alleviating the disease, condition, or syndrome, even if the disease, condition, or syndrome subsides. Therefore, the terms “treating,” “treat,” and “treatment” encompass both preventative (e.g., preventative) and palliative treatment. As is known in the art, it may be necessary to adjust for systemic and local delivery, age, weight, general health status, sex, diet, timing of administration, drug interactions, and severity of symptoms, and such adjustments will be determined by those skilled in the art through routine experiments.

[0113] "Subject" encompasses mammals such as humans, non-human primates, livestock, companion animals, and laboratory animals (e.g., rodents and lagomorphs). The term is intended to encompass both men and women.

[0114] "Patient" means any subject who is undergoing disease assessment, treatment, or experiencing a disease.

[0115] "Infant" means a child from the first month after birth to about one (1) year of age. As used herein, the term "newborn" means a child from birth to 28 days after birth. The term "premature infant" means an infant born after 20 weeks of gestation but before full term, typically weighing between about 500 g and about 2499 g at birth. "Very low birth weight infant" means an infant weighing less than 1500 g at birth.

[0116] "Child" means a person who has not yet reached the legal age of consent to receive treatment or research procedures. In some embodiments, the term means a person between birth and puberty.

[0117] "Adult" means a person who has reached the legal age (e.g., 18 years) in the relevant jurisdiction. In some embodiments, the term refers to any fully developed organism. In some embodiments, the term "adolescent" refers to a person under the age of 18 who has reached sexual maturity.

[0118] Engineered GAA Peptides

[0119] This disclosure provides engineered acid α-glucosidase peptides characterized by improved properties compared to wild-type acid α-glucosidase or reference engineered acid α-glucosidase peptides. The engineered acid α-glucosidase peptides described herein have been engineered to have improved activity, plasma stability, cellular uptake, lysosomal stability, and / or acid pH stability, etc. The engineered acid α-glucosidase peptides can be used for therapeutic applications, such as for treating symptoms associated with acid α-glucosidase deficiency.

[0120] In one aspect, this disclosure provides an engineered acidic α-glucosidase polypeptide or a bioactive fragment thereof, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NOs corresponding to even-numbered residues in SEQ ID NOs: 2, 12, and 14 to 754, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20 to 944 in SEQ ID NO: 12 or 2 or relative to the reference sequence corresponding to SEQ ID NO: 12 or 2.

[0121] In some embodiments, the engineered acidic α-glucosidase or its bioactive fragment comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 12 or 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 12 or 2.

[0122] In some embodiments, the engineered acidic α-glucosidase or its bioactive fragment comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 12, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or relative to the reference sequence corresponding to SEQ ID NO: 12.

[0123] In some embodiments, the engineered acidic α-glucosidase or its bioactive fragment comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO. in SEQ ID NO: 14 to 754, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2 or relative to the reference sequence corresponding to SEQ ID NO: 12 or 2.

[0124] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase comprises at least the amino acid positionsSubstitutions at 24, 28, 29, 39, 50, 62, 78, 87, 135, 150, 266, 267, 305, 437, 486, 522, 569, 670, 692, 711, 736, 750, 812, 830, 842, 871, 883, 894, 913 or 932 or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2 or relative to the reference sequence corresponding to SEQ ID NO: 12 or 2.

[0125] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase at least includes substituted or amino acid residues 24A / C / D / E / F / G / H / I / K / L / M / N / P / R / S / T / V / Y, 28A / C / D / E / F / G / H / K / L / P / Q / R / T / V / W, 29A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y, 39A / E / F / G / I / L / N / T, 50A / C / D / E / F / G / H / I / K / L / M / N / Q / R / S / T / W / Y, 62A / D / E / F / G / H / I / K / M / N / P / Q / S / T / V / Y, 78A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / V / W / Y, 87A / D / G / H / I / K / L / MN / Q / R / S / T / V / W, 135A / C / D / E / F / G / H / I / K / L / N / P / R / Y, 150T, 266A / D / E / H / K / Q / T, 267H / L / R / T / V, 305V, 437A / H / S, 486A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 522A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y, 569A / C / D / E / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y, 670A / D / E / F / G / H / I / K / L / M / N / Q / R / S / V / Y, 692A / C / D / E / F / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 711A / C / D / E / F / G / I / K / L / M / N / Q / R / S / T / V / W / Y, 736F / L, 750A / E / K / L / Q / R, 812A / D / G / S, 830D / E / F / G / H / L / M / N / Q / S / T / V / W / Y, 842A / C / D / E / F / G / H / K / L / M / N / Q / R / T / W,871A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 883A / F / Q, 894A / C / D / E / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 913E / F / H / I / K / M / N / Q / S / W, or 932C / D / E / G / H / K / L / M / N / P / Q / R / S / T / W / Y, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2, or relative to the reference sequence corresponding to SEQ ID NO: 12 or 2.

[0126] In some embodiments, the amino acid sequence of the engineered acid α-glucosidase includes at least the substitutions W24E / A / C / D / F / G / H / I / K / L / M / N / P / R / ST / V / Y, S28A / C / D / E / F / G / H / K / L / P / Q / R / T / V / W, T29A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y, Q39A / E / F / G / I / L / N / T, V50A / C / D / E / F / G / H / I / K / L / M / N / Q / R / S / T / W / Y, L62A / D / E / F / G / H / I / K / M / N / P / Q / S / T / V / Y, E78A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / V / W / Y, E87A / D / G / H / I / K / L / M / N / Q / R / S / T / V / W, Q135A / C / D / E / F / G / H / I / K / L / N / P / R / Y, S150T, N266A / D / E / H / K / Q / T, K267H / L / R / T / V, G437A / H / S, E486A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, V522A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y, T569A / C / D / E / G / H / I / K / L / Instruction Manual 15 / 115 Page 19 CN 121511299 AM / N / P / Q / R / S / V / W / Y, T670A / D / E / F / G / H / I / K / L / M / N / Q / R / S / V / Y, G692A / C / D / E / F / H / I / K / L / M / N / Q / R / S / T / V / W / Y, H711A / C / D / E / F / G / I / K / L / M / N / Q / R / S / T / V / W / Y, M736F / L, P750A / E / K / L / Q / R, E812A / D / G / S, K830D / E / F / G / H / L / M / N / Q / S / T / V / W / Y, S842A / C / D / E / F / G / H / K / L / M / N / Q / R / T / W, E871A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, H883A / F / Q, G894A / C / D / E / H / I / K / L / M / N / Q / R / S / T / V / W / Y, R913E / F / H / I / K / M / N / Q / S / W, A932C / D / E / G / H / K / L / M / N / P / Q / R / S / T / W / Y, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2, or relative to the reference sequence corresponding to SEQ ID NO: 12 or 2.

[0127] In some embodiments, the amino acid sequence of the engineered acid α-glucosidase includes at least the substituted or amino acid residues 24A / C / D / F / G / H / I / K / M / N / P / S / T / V / Y, 28A / C / D / E / F / G / H / K / Q / T / V / W, 29A / C / D / E / F / G / H / I / K / M / N / P / R / W / Y, 39A / E / F / G / I / L / N / T, 50A / C / D / E / F / H / I / K / M / N / R / S / T / W / Y, 62D / H / I / K / M / N / P / Q / Y, 78A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 87A / G / H / I / K / L / MN / Q / R / S / T / V / W, 135C / D / E / F / G / H / I / K / L / N / R / Y, 266A / D / E / H / K / Q, 267H / L / T / V, 305V, 437A / H, 486C / D / F / G / H / I / K / L / M / N / Q / R / S / V / W / Y, 522A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y, 569A / C / D / E / G / K / M / N / P / R / W, 670A / D / G / H / K / M / Y, 692A / D / E / H / K / L / M / N / T / W, 711D / E / I / K / M / N / Q / S / T / V / Y, 736F / L, 750E / K / L / Q / R, 812A / D / G / S, 830D / E / F / G / H / L / M / N / S / T / W / Y, 842A / C / D / F / H / K / L / M / N / Q / R / T / W, 871A / C / D / F / H / I / M / N / Q / T / V / W / Y, 883A / F / Q, 894A / D / E / H / I / K / L / M / N / S / T / V / W / Y, 913F / I / K / M / N / S or 932C / D / E / G / H / K / L / M / N / P / Q / R / W / Y, or combinations thereof, wherein the amino acid position is relative to the position corresponding to SEQ ID NO:The reference sequence of residues 20 to 944 of SEQ ID NO: 12 or the reference sequence corresponding to SEQ ID NO: 12.

[0128] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase at least includes substitutions for W24A / C / D / F / G / H / I / K / M / N / P / S / T / V / Y, S28A / C / D / E / F / G / H / K / Q / T / V / W, T29A / C / D / E / F / G / H / I / K / M / N / P / R / W / Y, Q39A / E / F / G / I / L / N / T, V50A / C / D / E / F / H / I / K / M / N / R / S / T / W / Y, L62D / H / I / K / M / N / P / Q / Y, E78A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, E87A / G / H / I / K / L / MN / Q / R / S / T / V / W, Q135C / D / E / F / G / H / I / K / L / N / R / Y, N266A / D / E / H / K / Q, K267H / L / T / V, L305V, G437A / H, E486C / D / F / G / H / I / K / L / M / N / Q / R / S / V / W / Y, V522A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y, T569A / C / D / E / G / K / M / N / P / R / W, T670A / D / G / H / K / M / Y, G692A / D / E / H / K / L / M / N / T / W, H711D / E / I / K / M / N / Q / S / T / V / Y, M736F / L, P750E / K / L / Q / R, E812A / D / G / S, K830D / E / F / G / H / L / M / N / S / T / W / Y, S842A / C / D / F / H / K / L / M / N / Q / R / T / W, E871A / C / D / F / H / I / M / N / Q / T / V / W / Y, H883A / F / Q, G894A / D / E / H / I / K / L / M / N / S / T / V / W / Y, R913F / I / K / M / N / S, or A932C / D / E / G / H / K / L / M / N / P / Q / R / W / Y, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or relative to the reference sequence corresponding to SEQ ID NO: 12.

[0129] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution at amino acid position 305. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid residue 305V. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution L305V.

[0130] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution at amino acid position 24. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid residue 24A / C / D / F / G / H / I / K / M / N / P / S / T / V / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid residue 24A / C / D / E / F / G / H / I / K / L / M / N / P / R / ST / V / Y. In some embodiments described on pages 16 / 115 of the specification (20 CN 121511299 A), the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution W24A / C / D / F / G / H / I / K / M / N / P / S / T / V / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution W24E / A / C / D / F / G / H / I / K / L / M / N / P / R / ST / V / Y.

[0131] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution at amino acid position 28. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 28A / C / D / E / F / G / H / K / Q / T / V / W. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 28A / C / D / E / F / G / H / K / L / P / Q / R / T / V / W. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution S28A / C / D / E / F / G / H / K / Q / T / V / W. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution S28A / C / D / E / F / G / H / K / L / P / Q / R / T / V / W.

[0132] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution at amino acid position 29. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 29A / C / D / E / F / G / H / I / K / M / N / P / R / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 29A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitutions for T29A / C / D / E / F / G / H / I / K / M / N / P / R / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitutions for...T29A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y.

[0133] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution at amino acid position 39. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid residue 39A / E / F / G / I / L / N / T. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution Q39A / E / F / G / I / L / N / T.

[0134] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution at amino acid position 50. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid residue 50A / C / D / E / F / H / I / K / M / N / R / S / T / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 50A / C / D / E / F / G / H / I / K / L / M / N / Q / R / S / T / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution V50A / C / D / E / F / H / I / K / M / N / R / S / T / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution V50A / C / D / E / F / G / H / I / K / L / M / N / Q / R / S / T / W / Y.

[0135] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution at amino acid position 62. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 62D / H / I / K / M / N / P / Q / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 62A / D / E / F / G / H / I / K / M / N / P / Q / S / T / V / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution L62D / H / I / K / M / N / P / Q / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution L62A / D / E / F / G / H / I / K / M / N / P / Q / S / T / V / Y.

[0136] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution at amino acid position 78. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 78A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y. In some embodiments, engineered acidic α-glucoseThe amino acid sequence of the glycosidase contains at least the substitution or amino acid residue 78A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / V / (Specification 17 / 115 pages 21 CN 121511299 AW / Y). In some embodiments, the amino acid sequence of the engineered acid α-glucosidase contains at least the substitution E78A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y. In some embodiments, the amino acid sequence of the engineered acid α-glucosidase contains at least the substitution E78A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / V / W / Y.

[0137] In some embodiments, the amino acid sequence of the engineered acid α-glucosidase contains at least the substitution at amino acid position 87. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 87A / G / H / I / K / L / MN / Q / R / S / T / V / W. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 87A / D / G / H / I / K / L / M / N / Q / R / S / T / V / W. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution E87A / G / H / I / K / L / MN / Q / R / S / T / V / W. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution E87A / D / G / H / I / K / L / M / N / Q / R / S / T / V / W.

[0138] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution at amino acid position 135. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 135C / D / E / F / G / H / I / K / L / N / R / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 135A / C / D / E / F / G / H / I / K / L / N / P / R / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution Q135C / D / E / F / G / H / I / K / L / N / R / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution Q135A / C / D / E / F / G / H / I / K / L / N / P / R / Y.

[0139] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution at amino acid position 150. In some embodiments, the amino acid sequence of the engineered acid α-glucosidase contains at least substitutions or amino groups.Amino acid residue 150T. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution S150T.

[0140] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution at amino acid position 266. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 266A / D / E / H / K / Q. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 266A / D / E / H / K / Q / T. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution N266A / D / E / H / K / Q / T. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution N266A / D / E / H / K / Q / T.

[0141] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution at amino acid position 267. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid residue 267H / L / T / V. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid residue 267H / L / R / T / V. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution K267H / L / T / V. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution K267H / L / R / T / V.

[0142] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution at amino acid position 437. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid residue 437H. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid residue 437A / H / S. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution G437H. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution G437A / H / S.

[0143] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution at amino acid position 486. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 486C / D / F / G / H / I / K / L / M / N / Q / R / S / V / W / Y. In some embodiments, the engineered acidic α-glucosidase...The amino acid sequence of the glycosidase contains at least the substitution or amino acid residue 486A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y. In some embodiments, the amino acid sequence of the engineered acid α-glucosidase contains at least the substitution E486C / D / F / G / H / I / K / L / M / N / Q / R / S / V / W / Y. In some embodiments, the amino acid sequence of the engineered acid α-glucosidase contains at least the substitution E486A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y.

[0144] In some embodiments, the amino acid sequence of the engineered acid α-glucosidase contains at least the substitution at amino acid position 522. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substituted amino acid residue 522A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substituted amino acid residue 522A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substituted V522A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substituted V522A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y.

[0145] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution at amino acid position 569. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid residue 569A / C / D / E / G / K / M / N / P / R / W. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid residue 569A / C / D / E / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution T569A / C / D / E / G / K / M / N / P / R / W. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution T569A / C / D / E / G / H / I / K / L / M / N / P / Q / R / S / V / W / Y.

[0146] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution at amino acid position 670. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid position 670.Amino acid residue 670A / D / G / H / K / M / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 670A / D / E / F / G / H / I / K / L / M / N / Q / R / S / V / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution T670A / D / G / H / K / M / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution T670A / D / E / F / G / H / I / K / L / M / N / Q / R / S / V / Y.

[0147] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution at amino acid position 692. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 692A / D / E / H / K / L / M / N / T / W. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 692A / C / D / F / H / I / K / L / M / N / Q / R / S / T / V / W / Y / E. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution G692A / D / E / H / K / L / M / N / T / W. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution G692A / C / D / E / F / H / I / K / L / M / N / Q / R / S / T / V / W / Y.

[0148] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution at amino acid position 711. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 711D / E / I / K / M / N / Q / S / T / V / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 711A / C / D / E / F / G / I / K / L / M / N / Q / R / S / T / V / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution H711D / E / I / K / M / N / Q / S / T / V / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution H711A / C / D / E / F / G / I / K / L / M / N / Q / R / S / T / V / W / Y.

[0149] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution at amino acid position 736. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 711A / C / D / E / F / G / I / K / L / M / N / Q / R / S / T / V / W / Y.Amino acid residue 736F / L. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution M736F / L.

[0150] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution at amino acid position 750. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 750E / K / L / Q / R. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 750A / E / K / L / Q / R. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution P750E / K / L / Q / R. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution P750A / E / K / L / Q / R.

[0151] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution at amino acid position 812. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid residue 812A / D / G / S. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution E812A / D / G / S.

[0152] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution at amino acid position 830. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid residue 830D / E / F / G / H / L / M / N / S / T / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid residue 830D / E / F / G / H / L / M / N / Q / S / T / V / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution K830D / E / F / G / H / L / M / N / S / T / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution K830D / E / F / G / H / L / M / N / Q / S / T / V / W / Y.

[0153] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution at amino acid position 842. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 842A / C / D / F / H / K / L / M / N / Q / R / T / W. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 842A / C / D / E / F / G / H / K / L / M / N / Q / R / T / W. In some embodimentsFor example, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution S842A / C / D / F / H / K / L / M / N / Q / R / T / W. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution S842A / C / D / E / F / G / H / K / L / M / N / Q / R / T / W.

[0154] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution at amino acid position 871. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 871A / C / D / F / H / I / M / N / Q / T / V / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution or amino acid residue 871A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution of E871A / C / D / F / H / I / M / N / Q / T / V / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution of E871A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y.

[0155] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution at amino acid position 20 / 115 page 24 CN 121511299 A 883. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution of amino acid residue 883A / F / Q. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substitution of H883A / F / Q.

[0156] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution at amino acid position 894. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid residue 894A / D / E / H / I / K / L / M / N / S / T / V / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid residue 894A / C / D / E / H / I / K / L / M / N / Q / R / S / T / V / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution G894A / D / E / H / I / K / L / M / N / S / T / V / W / Y. In some embodiments, the amino acid sequence of the engineered acid α-glucosidase contains at least the substituted G894A / C / D / E / H / I / K / L / M / N / Q / R / S / T / V / W / Y.

[0157] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution at amino acid position 913. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid residue 913F / I / K / M / N / S. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution or amino acid residue 913E / F / H / I / K / M / N / Q / S / W. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution R913F / I / K / M / N / S. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution R913E / F / H / I / K / M / N / Q / S / W.

[0158] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least a substitution at amino acid position 932. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substituted amino acid residue 932C / D / E / G / H / K / L / M / N / P / Q / R / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substituted amino acid residue 932C / D / E / G / H / K / L / M / N / P / Q / R / S / T / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substituted A932C / D / E / G / H / K / L / M / N / P / Q / R / W / Y. In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the substituted A932C / D / E / G / H / K / L / M / N / P / Q / R / S / T / W / Y.

[0159] In some embodiments, the engineered acidic α-glucosidase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with an engineered acidic α-glucosidase having the substitutions or substitution sets shown in Table 3-1, wherein the amino acid position is relative to a reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2 or relative to a reference sequence corresponding to SEQ ID NO: 12 or 2.

[0160] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase comprises at least the substitutions at the amino acid positions shown in Table 3-1, wherein the amino acid position is relative to a reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2 or relative to a reference sequence corresponding to SEQ ID NO: 12 or 2.

[0161] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least one substitution as shown in Table 3-1, wherein the amino acid position is relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2, or relative to the reference sequence corresponding to SEQ ID NO: 12 or 2.

[0162] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase contains at least the set of substitutions as shown in Table 3-1, wherein the amino acid position is relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0163] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase includes at least the amino acid positions 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 305 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 871 / 883 / 894 / 913 / 932、28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842S / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 842 / 871 / 883 / 894 / 913 / 932、 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 or 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / The substitution set at position 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812S / 830 / 842 / 871 / 883 / 894 / 913 / 932, page 22 / 115 of the specification, CN 121511299 A, wherein the amino acid position is relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the specific amino acid substitution at each position in the substitution set is selected from the substitutions described herein for each amino acid position.

[0164] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase at least includes the substitution set 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 305V / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135P / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486Q / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842E / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78G / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894S / 913R / 932A、24W / 28S / 29A / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62S / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932R、24W / 28A / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569A / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569R / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842W / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29C / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267L / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87Q / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62D / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62H / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135R / 150S / 266N / 23 / 115 pages of the specification 27 CN 121511299 A 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871A / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50S / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569G / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692R / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830N / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894R / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267T / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24E / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135G / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692V / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87R / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913Q / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711L / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87L / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87V / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78Y / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569C / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87W / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670A / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569Q / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62K / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / , 24 / 115 pages of the specification, 28 CN 121511299 A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569Q / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62K / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 69Z / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894C / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569H / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711V / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522S / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24F / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569M / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711S / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24Y / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87G / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78I / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78R / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24C / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78W / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569K / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24S / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135L / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842R / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894Y / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842T / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894T / 913R / 932A24W / 28Q / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266H / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62P / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39L / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486D / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711G / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24N / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711W / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871R / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 75Q / 812E / 830K / 842S / 871E / 883H / 89LG / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522W / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894L / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 26 / 115 pages of the specification 30 CN 121511299 A 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883A / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522G / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522L / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670V / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62G / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 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670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486L / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, Page 27 / 115 of the specification 31 CN 121511299 A24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913E / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711R / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39G / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24R / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 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/ 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486N / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894V / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830D / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894W / 913R / 932A, Description 30 / 115 pages 34 CN 121511299 A 24W / 28S / 29T / 39Q / 50V / 62L / 78V 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267K / 437G / 486E / 522T / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24K / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871K / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750E / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830T / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50I / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932E、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266Q / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, Description, page 33 / 115, 37 CN 121511299 A 24W / 28S / 29T / 39A / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24T / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 8(continued on next page)30K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871T / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842M / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711T / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266K / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486F / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50M / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 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/ 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670K / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692A / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50T / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842H / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692M / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29Q / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894D / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913F / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670D / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62M / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486C / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A24W / 28S / 29T / 39Q / 50V / 62L / 78T / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267H / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29I / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 35 / 115 pages of the specification 39 CN 12151,1299 A 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24H / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 并486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135I / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / ⑧12E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692H / 711H / 736M / It should be noted that there seems to be some unclear or incorrect content in the original text (such as "并486E" and "⑧12E"), which may affect the accuracy of a perfect translation. This translation attempts to handle the text as accurately as possible based on the existing content.750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28H / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913N / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842D / 871E / 883H / 894G / 913R / 932A、 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522K / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894H / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78K / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39F / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871W / 883H / 894G / 913R / 932A、 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Y / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842Q / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29H / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78Q / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522D / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932Y, 24W / 28S / 29V / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 750P / 812E / 830K / 842S / 871E / 883H / 894I / 913R / 932A、24W / 28S / 29T / 39N / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266E / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78C / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932P、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932D、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830Y / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135K / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692D / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28G / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711N / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486W / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50D / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486A / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894N / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913K / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871M / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39E / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932N、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932M、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913M / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569W / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486S / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711I / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711C / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50W / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522C / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R, 24W / 28S / 29W / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812G / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 437G / 486E / 522M / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670N / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29M / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486G / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932K、24W / 28S / 29T / 39Q / 50V / 62Q / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830S / 842S / 871E / 883H / 894G / 913R / 932A、 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812S / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830G / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486M / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711K / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932H、 24W / 28S / 29P / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692W / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29Y / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692C / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29G / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A、 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812D / 830K / 842S / 871E / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932C、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871I / 883H / 894G / 913R / 932A、24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692Y / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750Q / 812E / 830K / 842S / 871E / 883H / 894G / 913R / 932A, Instruction Manual, 39 / 115 pages, 43 CN 121511299 A 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830H / 842S / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842C / 871E / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871S / 883H / 894G / 913R / 932A, 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871C / 883H / 894G / 913R / 932A or 24W / 28S / 29T / 39Q / 50V / 62L / 78E / 87E / 135Q / 150S / 266N / 267K / 437G / 486E / 522V / 569T / 670T / 692G / 711H / 736M / 750P / 812E / 830K / 842S / 871E / 883H / 894G / 913W / 932A, where the amino acid position is relative to the SEQ ID NO: The reference sequence of residues 20 to 944 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0165] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase comprises SEQ ID NO: 14 to 944.Residues 20 to 944 of the even-numbered SEQ ID NO. in SEQ ID NO: 754, or containing the even-numbered SEQ ID NO. in SEQ ID NO: 14 to 754.

[0166] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase comprises SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452,454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, The amino acid sequence may contain residues 20 to 944 of 682, 684, 686, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, or 754. In some embodiments, the amino acid sequence may optionally have 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid insertions, deletions, or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, or up to 5 amino acid insertions, deletions, or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, or up to 5 amino acid substitutions. In some embodiments, the amino acid substitutions include non-conservative or conservative substitutions. In some embodiments, the amino acid substitutions include conservative substitutions. In some embodiments, the variants and examples disclosed herein provide guidance on non-conservative and conservative substitutions.

[0167] In some embodiments, the amino acid sequence of the engineered acidic α-glucosidase comprises SEQ ID NO: 14,16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60、62、64、 66、68、70、72、74、76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110、 112、114、116、118、120、122、124、126、128、130、132、134、136、138、140、142、144、146、148、 150、152、154、156、158、160、162、164、166、168、170、172、174、176、178、180、182、184、186、 188、190、192、194、196、198、200、202、204、206、208、210、212、214、216、218、220、222、224、 226、228、230、232、234、236、238、240、242、244、246、248、250、252、254、256、258、260、262、 264、266、268、270、272、274、276、278、280、282、284、286、288、290、292、294、296、298、300、 302、304、306、308、310、312、314、316、318、320、322、324、326、328、330、332、334、336、338、 340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、370、372、374、376、 378、380、382、384、386、388、390、392、394、396、398、400、402、404、406、408、410、412、414、 416、418、420、422、424、426、428、430、432、434、436、438、440、442、444、446、448、450、452、 454、456、458、460、462、464、466、468、470、472、474、476、478、480、482、484、486、488、490、 492、494、496、498、500、502、504、506、508、510、512、514、516、518、520、522、524、526、528、530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752 or 754. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid insertions, deletions, or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, or up to 5 amino acid insertions, deletions, or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, or up to 5 amino acid substitutions. In some embodiments, the amino acid substitutions include non-conservative or conservative substitutions. In some embodiments, the amino acid substitutions include conservative substitutions. In some embodiments, the variants and examples disclosed herein provide guidance on non-conservative and conservative substitutions.

[0168] In some embodiments, the engineered acidic α-glucosidase of this disclosure has acidic α-glucosidase activity. In some embodiments, the engineered acidic α-glucosidase has acidic α-glucosidase activity, and at least one improved or enhanced property compared to a reference acidic α-glucosidase.

[0169] In some embodiments, the engineered acidic α-glucosidase has higher thermal stability compared to a reference acidic α-glucosidase.

[0170] In some embodiments, the engineered acidic α-glucosidase has higher stability at pH 7 (e.g., at neutral pH) compared to a reference acidic α-glucosidase.

[0171] In some embodiments, engineered acidic α-glucosidase exhibits higher stability at acidic pH (particularly around pH 4.4) compared to a reference acidic α-glucosidase.

[0172] In some embodiments, engineered acidic α-glucosidase exhibits higher expression compared to a reference acidic α-glucosidase.

[0173] In some embodiments, engineered acidic α-glucosidase exhibits higher stability in lysosomes compared to a reference acidic α-glucosidase.

[0174] In some embodiments, engineered acidic α-glucosidase exhibits higher stability in plasma (particularly human plasma) compared to a reference acidic α-glucosidase.

[0175] In some embodiments, engineered acidic α-glucosidase is more readily absorbed by human cells compared to a reference acidic α-glucosidase.

[0176] In some embodiments, the engineered acidic α-glucosidase exhibits higher enzymatic activity in cell lysates compared to a reference acidic α-glucosidase.

[0177] In some embodiments, the engineered acidic α-glucosidase exhibits reduced immunogenicity compared to a reference acidic α-glucosidase.

[0178] In some embodiments, the reference acidic α-glucosidase has a sequence corresponding to residues 20 to 944 of SEQ ID NO: 2 or 12, or a sequence corresponding to SEQ ID NO: 2 or 12. In some embodiments, the reference acidic α-glucosidase has a sequence corresponding to residues 20 to 944 of SEQ ID NO: 2, or a sequence corresponding to SEQ ID NO: 2. In some embodiments, the reference acidic α-glucosidase has a sequence corresponding to residues 20 to 944 of SEQ ID NO: 12, or a sequence corresponding to SEQ ID NO: 12. Exemplary improved properties are provided in the examples.

[0179] In some embodiments, compared with a reference acidic α-glucosidase, the engineered acidic α-glucosidase exhibits at least one improved property selected from the following: i) enhanced catalytic activity; ii) improved tolerance to pH 7; iii) improved tolerance to pH 4.4; iv) improved stability in lysosomes; v) increased expression; vi) increased uptake into cells; vii) increased enzymatic activity in cell lysates; viii) improved stability in plasma / serum; and ix) decreased immunogenicity; or a combination of any one of i), ii), iii), iv), v), vi), vii), viii), and ix). In some embodiments, the reference acidic α-glucosidase has a sequence corresponding to residues 20 to 944 of SEQ ID NO: 2 or 12, or corresponding to SEQ ID NO:The sequence is 2 or 12. In some embodiments, the reference acidic α-glucosidase has a sequence of residues 20 to 944 corresponding to SEQ ID NO: 12, or a sequence corresponding to SEQ ID NO: 12.

[0180] In some embodiments, the engineered acidic α-glucosidase exhibits reduced immunogenicity compared to the reference acidic α-glucosidase having a sequence of residues 20 to 944 corresponding to SEQ ID NO: 2 or 12, or a sequence corresponding to SEQ ID NO: 2 or 12. In some embodiments, the engineered acidic α-glucosidase exhibits a total immunogenicity score (TIS) reduction of more than 10, more than 100, or more than 200 compared to the reference acidic α-glucosidase of SEQ ID NO: 2. In some embodiments, the engineered acidic α-glucosidase exhibits an immunogenicity hit count (IHC) reduction of more than 2, more than 5, or more than 20 compared to the reference acidic α-glucosidase of SEQ ID NO: 2. In some embodiments, the engineered acidic α-glucosidase exhibits a total immunogenicity score (TIS) reduction of more than 10, more than 100, or more than 200 compared to the reference acidic α-glucosidase of SEQ ID NO: 12. In some embodiments, the engineered acidic α-glucosidase exhibits an immunogenicity hit count (IHC) reduction of more than 2, more than 5, or more than 20 compared to the reference acidic α-glucosidase of SEQ ID NO: 12. Exemplary engineered acidic α-glucosidase peptides exhibiting reduced immunogenicity based on TIS and / or IHC can be selected from the engineered acidic α-glucosidases shown in Table 4-1 of the examples.

[0181] In some embodiments, in MHC-related peptide proteomics (MAPP) assays as provided in the examples, engineered acidic α-glucosidases exhibit a reduced number of sequence regions presented by antigen-presenting cells and / or a decreased peptide presentation frequency compared to a reference acidic α-glucosidase polypeptide (particularly the reference acidic α-glucosidase polypeptide corresponding to SEQ ID NO: 2). Specification 42 / 115 pages 46 CN 121511299 A

[0182] In some embodiments, the engineered acidic α-glucosidase described herein comprises a precursor polypeptide of the engineered acidic α-glucosidase. In some embodiments, the precursor polypeptide of the engineered acidic α-glucosidase comprises a eukaryotic or synthetic signal peptide sequence. In some embodiments, the signal peptide of the precursor polypeptide of the engineered acidic α-glucosidase comprises a mouse or human signal peptide sequence. In some embodiments, the signal peptide comprises a sequence containing residues 1 to 19 of SEQ ID NO: 2 or 12.

[0183] In some embodiments, the engineered acidic α-glucosidase comprises the engineered acidic α-glucose described herein.The propeptide of an acid α-glucosidase. In some embodiments, the propeptide of an engineered acid α-glucosidase lacks a signal sequence. In some embodiments, the propeptide of an engineered acid α-glucosidase comprises residues 20 to 944 of the engineered acid α-glucosidase described herein.

[0184] In some embodiments, the engineered acid α-glucosidase is a mature form of the engineered acid α-glucosidase polypeptide described herein. In some embodiments, the mature form of the engineered acid α-glucosidase is a secretory form of the engineered acid α-glucosidase.

[0185] In some embodiments, the engineered acid α-glucosidase further comprises a fusion polypeptide. In some embodiments, the engineered acid α-glucosidase polypeptide described herein may be fused with a variety of polypeptide sequences, such as polypeptide tags that, by way of example but not limitation, can be used for detection and / or purification. In some embodiments, the fusion protein of the engineered acid α-glucosidase polypeptide comprises a glycine-histidine or histidine tag (His tag). In some embodiments, the fusion protein of the engineered acidic α-glucosidase peptide includes an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In some embodiments, the fusion protein of the engineered acidic α-glucosidase peptide includes a GST, SUMO, Strep, MBP, or GFP tag. In some embodiments, fusion occurs at the amino (N-) terminus of the engineered acidic α-glucosidase peptide. In some embodiments, fusion occurs at the carboxyl (C-) terminus of the engineered acidic α-glucosidase peptide. In some embodiments, the fusion peptide is inserted after the signal sequence and before the engineered acidic α-glucosidase peptide to achieve expression and secretion of a peptide comprising the fusion peptide (e.g., a peptide tag) and the peptide.

[0186] In some embodiments, the engineered acidic α-glucosidase is an isolated or purified formulation. In some embodiments, the engineered acidic α-glucosidase is purified from a mixture (e.g., from cells or culture medium) using any one or more known techniques for protein purification (including but not limited to lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography).

[0187] Chromatographic techniques used for the separation and purification of engineered acidic α-glucosidase peptides include, but are not limited to, reversed-phase chromatography, high-performance liquid chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, gel electrophoresis, and affinity chromatography. The conditions used to purify a particular protein depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, and molecular shape, and will be apparent to those skilled in the art. In some embodiments, affinity techniques can be used to separate peptides. For affinity chromatography purification, any antibody that specifically binds to the target acidic α-glucosidase peptide can be used.Used to generate antibodies, various host animals (including, but not limited to, rabbits, mice, rats, camels, etc.) are immunized by injection of engineered acidic α-glucosidase peptides or fragments thereof.

[0188] In some embodiments, this disclosure further provides functional or bioactive fragments of the engineered acidic α-glucosidase peptides described herein. Thus, for each embodiment of the engineered acidic α-glucosidase herein, a functional or bioactive fragment of the engineered acidic α-glucosidase is provided herein. In some embodiments, the functional or bioactive fragment of the engineered acidic α-glucosidase comprises at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the activity of the acidic α-glucosidase peptide from which it is derived (i.e., the parental acidic α-glucosidase). Specification 43 / 115 pages 47 CN 121511299 A

[0189] In some embodiments, the functional or bioactive fragment of the engineered acid α-glucosidase herein comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parental sequence of the engineered acid α-glucosidase. In some embodiments, the functional or bioactive fragment comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parental sequence of the acid α-glucosidase. In some embodiments, the functional fragment will be truncated to fewer than 5, 10, 15, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 amino acids.

[0190] In some embodiments, the functional or bioactive fragment of the engineered acid α-glucosidase polypeptide described herein includes at least one substitution or set of substitutions in the amino acid sequence of the engineered acid α-glucosidase described herein. Thus, in some embodiments, one or more functional fragments or one or more bioactive fragments of the engineered acid α-glucosidase exhibit enhanced or improved properties associated with the substitutions or set of substitutions in the parental acid α-glucosidase.

[0191] Polynucleotide encoding recombinant polypeptide, expression vector, and host cell

[0192] In another aspect, this disclosure provides recombinant polynucleotides encoding the engineered acid α-glucosidase polypeptide described herein. In some embodiments, the polynucleotide is operatively linked to one or more heterologous or homologous regulatory sequences that control gene expression to form a recombinant polynucleotide capable of expressing a polypeptide. This may include a polynucleotide encoding an engineered acid.Expression constructs of heterologous polynucleotides of acid α-glucosidase polypeptides are introduced into suitable host cells to express the corresponding engineered acid α-glucosidase polypeptide.

[0193] It will be apparent to those skilled in the art that the availability of protein sequences and knowledge of codons corresponding to various amino acids provide a description of all polynucleotides capable of encoding the target polypeptide. The degeneracy of the genetic code (where the same amino acid is encoded by substitutional or synonymous codons) enables the synthesis of an extremely large number of nucleic acids, all of which encode engineered acid α-glucosidase polypeptides. Thus, those skilled in the art, knowing a particular amino acid sequence, can synthesize any number of different nucleic acids simply by modifying the sequence of one or more codons in a manner that does not alter the amino acid sequence of the protein. In this regard, this disclosure specifically contemplates encoding each possible polynucleotide variant of the engineered acid α-glucosidase polypeptide described herein by means of possible codon selection combinations, and all such variants should be considered as specifically disclosed for any polypeptide described herein (including the engineered acid α-glucosidase variants provided in the examples).

[0194] In some embodiments, the recombinant polynucleotide is codon-optimized. In other words, the codons are preferably selected to suit the host cell in which the protein is produced. For example, preferred codons used in bacteria are used for expression in bacteria, preferred codons used in fungi are typically used for expression in fungi, and preferred codons used in mammals are used for expression in mammals and mammalian cells. In some embodiments, the codon-optimized polynucleotide encoding the engineered acid α-glucosidase 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. In some embodiments, this disclosure provides recombinant polynucleotide sequences in which the codons are optimized for expression in human cells or tissues.

[0195] As described above, it should be understood that this disclosure provides recombinant polynucleotides encoding each of the engineered acid α-glucosidase polypeptides described herein. Therefore, by way of example but not limitation, in some embodiments, the recombinant polynucleotide of this disclosure comprises a polynucleotide sequence encoding an engineered acidic α-glucosidase or a bioactive fragment thereof, the engineered acidic α-glucosidase or bioactive fragment thereof comprising a reference sequence of residues 20 to 944 corresponding to even-numbered SEQ ID NOs in SEQ ID NOs: 2, 12 and 14 to 754, or SEQ ID NOs corresponding to even-numbered SEQ ID NOs in SEQ ID NOs: 2, 12 and 14 to 754.An amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher sequence identity, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2, or relative to the reference sequence corresponding to SEQ ID NO: 12 or 2.

[0196] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acidic α-glucosidase comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 12 or 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2, or relative to the reference sequence corresponding to SEQ ID NO: 12 or 2.

[0197] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acidic α-glucosidase comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 12, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or relative to the reference sequence corresponding to SEQ ID NO: 12.

[0198] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acidic α-glucosidase comprising a reference sequence of residues 20 to 944 of SEQ ID NO. corresponding to even-numbered SEQ ID NO. in SEQ ID NO: 14 to 754, or a reference sequence of residues 20 to 944 of SEQ ID NO. corresponding to even-numbered SEQ ID NO: 14 to 754.The reference sequence of NO. has an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher sequence identity, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2, or relative to the reference sequence corresponding to SEQ ID NO: 12 or 2.

[0199] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acidic α-glucosidase comprising an amino acid sequence comprising at least substitutions at amino acid positions 24, 28, 29, 39, 50, 62, 78, 87, 135, 150, 266, 267, 305, 437, 486, 522, 569, 670, 692, 711, 736, 750, 812, 830, 842, 871, 883, 894, 913, or 932 or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or relative to the reference sequence corresponding to SEQ ID NO: 12.

[0200] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 305.

[0201] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 24.

[0202] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 28.

[0203] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 29.

[0204] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence including at least a substitution at amino acid position 39.

[0205] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence including at least a substitution at amino acid position 50.

[0206] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 62.

[0207] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 78.

[0208] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 87.

[0209] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 135.

[0210] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 150.

[0211] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 266.

[0212] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 267.

[0213] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 437.

[0214] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 486.

[0215] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 522.

[0216] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 569.

[0217] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 670.

[0218] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 692.

[0219] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 711.

[0220] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 736.

[0221] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 750.

[0222] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 812.

[0223] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 830.

[0224] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 842. Specification 46 / 115 pages 50 CN 121511299 A

[0225] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 305.

[0226] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 883.

[0227] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 894.

[0228] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 913.

[0229] In some embodiments, the recombinant polynucleotide encodes an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence containing at least a substitution at amino acid position 913.α-Glucosidase comprises an amino acid sequence that includes at least a substitution at amino acid position 932.

[0230] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acidic α-glucosidase comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with an engineered acidic α-glucosidase having the substitutions or substitution sets shown in Table 3-1, wherein the amino acid position is relative to a reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2 or relative to a reference sequence corresponding to SEQ ID NO: 12 or 2.

[0231] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acidic α-glucosidase comprising an amino acid sequence comprising at least one substitution as shown in Table 3-1, wherein the amino acid position is relative to a reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2, or relative to a reference sequence corresponding to SEQ ID NO: 12 or 2.

[0232] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acidic α-glucosidase comprising an amino acid sequence comprising at least one set of substitutions as shown in Table 3-1, wherein the amino acid position is relative to a reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2, or relative to a reference sequence corresponding to SEQ ID NO: 12 or 2.

[0233] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence comprising at least the amino acid positions 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 305 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 871 / 883 / 894 / 913 / 932、28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / Instruction Manual 47 / 115 pages 51 CN 121511299 A 486 / 522 / 569 / 670 / 692 / 711 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842S / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932、24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 / 932, 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812 / 830 / 842 / 871 / 883 / 894 / 913 or 24 / 28 / 29 / 39 / 50 / 62 / 78 / 87 / 135 / 150 / 266 / 267 / Substitution at 437 / 486 / 522 / 569 / 670 / 692 / 711 / 736 / 750 / 812S / 830 / 842 / 871 / 883 / 894 / 913 / 932Set, wherein the position is relative to SEQ ID NO: 2.

[0234] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acidic α-glucosidase comprising an amino acid sequence comprising residues 20 to 944 of even-numbered SEQ ID NOs in SEQ ID NOs: 14 to 754, or comprising even-numbered SEQ ID NOs in SEQ ID NOs: 14 to 754.

[0235] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acidic α-glucosidase, the engineered acidic α-glucosidase comprising an amino acid sequence comprising SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112. 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, Instruction Manual 48 / 115 pages 52 CN 121511299 A 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340,342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, Residues 20 to 944 of 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, or 754.

[0236] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered acidic α-glucosidase comprising an amino acid sequence comprising SEQ ID NO: 14, 16, ...18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60、62、64、66、 68、70、72、74、76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110、112、 114、116、118、120、122、124、126、128、130、132、134、136、138、140、142、144、146、148、150、 152、154、156、158、160、162、164、166、168、170、172、174、176、178、180、182、184、186、188、 190、192、194、196、198、200、202、204、206、208、210、212、214、216、218、220、222、224、226、 228、230、232、234、236、238、240、242、244、246、248、250、252、254、256、258、260、262、264、 266、268、270、272、274、276、278、280、282、284、286、288、290、292、294、296、298、300、302、 304、306、308、310、312、314、316、318、320、322、324、326、328、330、332、334、336、338、340、 342、344、346、348、350、352、354、356、358、360、362、364、366、368、370、372、374、376、378、 380、382、384、386、388、390、392、394、396、398、400、402、404、406、408、410、412、414、416、 418、420、422、424、426、428、430、432、434、436、438、440、442、444、446、448、450、452、454、 456、458、460、462、464、466、468、470、472、474、476、478、480、482、484、486、488、490、492、 494、496、498、500、502、504、506、508、510、512、514、516、518、520、522、524、526、528、530、532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752 or 754.

[0237] In some embodiments, the recombinant polynucleotide comprises a reference polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or higher sequence identity with ...93%, 94%, 95%, 96%, 97% or higher sequence identity with a reference polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 93%, 94%, 95%, 96%, 97% or higher sequence identity with a reference polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 93%, 94%, 95%, 96%, 97% or higher sequence identity with a reference polynucleotide sequence having at least 70%, 75%, 80%, 81%, 92%, 93%, 94%, 95%, 96%, 97% or higher sequence identity with a reference polynucleotide sequence having at least 70%, 75%, 80%, 81%, 92%, 93%, 94%, 95%, 96%, 97% or higher sequence identity with a reference polynucleotide sequence having at least 70%, 75%, 80%, 81%, 92%, 93%, 9

[0238] In some embodiments, the recombinant polynucleotide comprises: a polynucleotide sequence comprising nucleotide residues 58 to 2832 of SEQ ID NO. with odd numbers from SEQ ID NO: 13 to 753; or a polynucleotide sequence comprising SEQ ID NO. with odd numbers from SEQ ID NO: 13 to 753.

[0239] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising SEQ ID NO:13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55、57、59、 61、63、65、67、69、71、73、75、77、79、81、83、85、87、89、91、93、95、97、99、101、103、105、107、 109、111、113、115、117、119、121、123、125、127、129、131、133、135、137、139、141、143、145、 147、149、151、153、155、157、159、161、163、165、167、169、171、173、175、177、179、181、183、 185、187、189、191、193、195、197、199、201、203、205、207、209、211、213、215、217、219、221、 223、225、227、229、231、233、235、237、239、241、243、245、247、249、251、253、255、257、259、 261、263、265、267、269、271、273、275、277、279、281、283、285、287、289、291、293、295、297、 299、301、303、305、307、309、311、313、315、317、319、321、323、325、327、329、331、333、335、 337、339、341、343、345、347、349、351、353、355、357、359、361、363、365、367、369、371、373、 375、377、379、381、383、385、387、389、391、393、395、397、399、401、403、405、407、409、411、 413、415、417、419、421、423、425、427、429、431、433、435、437、439、441、443、445、447、449、 451、453、455、457、459、461、463、465、467、469、471、473、475、477、479、481、483、485、487、 489、491、493、495、497、499、501、503、505、507、509、511、513、515、517、519、521、523、525、527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, Nucleotide residues 58 to 2832 of 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, or 753.

[0240] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, Instruction manual, 50 / 115 pages, 54 CN 121511299 A 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715,717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, or 753.

[0241] In some embodiments, the recombinant polynucleotides encoding any engineered acidic α-glucosidase polypeptides provided herein are manipulated in a variety of ways to provide expression of the encoded polypeptide. In some embodiments, the polynucleotide encoding the 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 polypeptide. In some embodiments, the control sequences include, but are not limited to, promoters, Kozak sequences, leader sequences, polyadenylated sequences, peptogen sequences, signal peptide sequences, DNA-based regulatory elements for gene therapy retention, and transcription terminators. Depending on the expression vector, it may be desirable or necessary to manipulate the isolated polynucleotide prior to inserting it into the vector. Techniques for modifying polynucleotide and nucleic acid sequences using recombinant DNA methods are well known in the art.

[0242] In some embodiments, a suitable promoter may be selected based on the host cell used for expression. For bacterial host cells, suitable promoters for guiding the transcription of the nucleic acid constructs of this disclosure include, but are not limited to: promoters derived from the *Escherichia coli* lactose operon, the *Streptomyces agarase* gene (dagA), the *Bacillus subtilis* fructan sucrase gene (sacB), the *Bacillus licheniformis* α-amylase gene (amyL), the *Bacillus thermophilus* maltose amylase gene (amyM), the *Bacillus amyloliquefaciens* α-amylase gene (amyQ), the *Bacillus licheniformis* penicillinase gene (penP), the *Bacillus subtilis* xylA and xylB genes, and prokaryotic β-lactamase genes (see, for example, Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA, 1978, 75:3727–3731); and the tac promoter (see, for example, DeBoer et al., Proc. Natl. Acad. Sci. USA, 1983, 80:21–25). Exemplary promoters for use in filamentous fungal host cells include, but are not limited to: promoters derived from the genes of *Aspergillus oryzae* TAKA amylase, *Rhizopus oryzae* aspartic protease, *Aspergillus niger* neutral α-amylase, *Aspergillus niger* acid-stable α-amylase, *Aspergillus niger* or *Aspergillus awamori* glucosylamylase (glaA), *Rhizopus oryzae* lipase, *Aspergillus oryzae* alkaline protease, *Aspergillus oryzae* triose phosphate isomerase, *Aspergillus nidus* acetamipridase, and *Fusarium oxysporum* trypsin-like protease (see, for example, WO 96 / 00787); and the NA2-tpi promoter (a hybrid of the promoters from the genes of *Aspergillus niger* neutral α-amylase and *Aspergillus oryzae* triose phosphate isomerase); and their mutant, truncated, and hybrid promoters.Example yeast cell promoters may be derived from the genes of *Saccharomyces cerevisiae* enolase (ENO-1), *Saccharomyces cerevisiae* galactokinase (GAL1), *Saccharomyces cerevisiae* alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and *Saccharomyces cerevisiae* 3-phosphate glycerate kinase. Other useful promoters for yeast host cells are known in the art (see, for example, Romanos et al., Yeast, 1992, 8:423-488). Exemplary promoters for use in mammalian cells include, but are not limited to, promoters derived from: cytomegalovirus (CMV), chicken β-actin promoter fused with a CMV enhancer, simian virus 40 (SV40), *Homo sapiens* phosphoglycerate kinase, β-actin, elongation factor-1a, or glyceraldehyde-3-phosphate dehydrogenase, or chicken β-actin.

[0243] In some embodiments, the control sequence is a suitable transcription terminator sequence (a sequence recognized by the host cell to terminate transcription). The terminator sequence is operatively linked to the 3' end of a nucleic acid sequence encoding a polypeptide. Any terminator that functions in a selected host cell can be used in this invention. For bacterial expression, the transcription terminator can be a Rho-dependent terminator that depends on the Rho transcription factor, or a Rho-independent or intrinsic terminator that does not require the transcription factor. Exemplary bacterial transcription terminators are described in the following literature: Peters et al., J Mol Biol., 2011, 412(5):793-813. Specification 51 / 115 pages 55 CN 121511299 A Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes of Aspergillus oryzae TAKA amylase, Aspergillus niger glucosylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-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, for example, Romanos et al., ibid.). Exemplary terminators for mammalian cells include, but are not limited to, those derived from cytomegalovirus (CMV), simian vacuolating virus 40 (SV40), human growth hormone (hGH), bovine growth hormone (BGH), and those derived from human or rabbit β-globulin.

[0244] In some embodiments, the control sequence is a suitable leader sequence, 5'-cap modification, 5' UTR, 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 uncapping. The leader sequence is operatively linked to the 5' end of a nucleic acid sequence encoding a polypeptide.Use any leader sequence that functions in a selected host cell. 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 α factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP). Suitable leaders for mammalian host cells include, but are not limited to, 5'-UTR elements present in orthopoxvirus mRNA.

[0245] In some embodiments, the control sequence comprises a 3' untranslated nucleic acid region and a polyadenylated tail nucleic acid sequence operatively linked to the 3' end of a protein-coding nucleic acid sequence, which mediates binding to proteins involved in mRNA transport and translation, as well as the mRNA half-life. Any polyadenylated sequence and 3' UTR that function in a selected host cell can be used in this invention. Exemplary polyadenylated sequences for filamentous fungal host cells include, but are not limited to, those from the genes of Aspergillus oryzae TAKA amylase, Aspergillus niger glucosylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger α-glucosidase. Useful polyadenylated sequences for yeast host cells are also known in the art (see, for example, Guo and Sherman, Mol. Cell. Biol., 1995, 15:5983-5990). Useful polyadenylated and 3' UTR sequences for mammalian host cells include, but are not limited to, the 3'-UTRs of α- and β-globulin mRNAs, which carry several sequence elements that increase mRNA stability and translation.

[0246] In some embodiments, the control sequence is a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide and guides the encoded polypeptide into the cell's secretory pathway. The 5' end of the coding sequence of a nucleic acid sequence may inherently contain a signal peptide coding region, which is naturally linked within the translation reading frame to a fragment encoding the coding region of a secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. Any signal peptide coding region that directs the expressed polypeptide to the secretory pathway of a selected host cell can be used for the expression of the engineered acid α-glucosidase polypeptide provided herein. Effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, those derived from the genes of Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Rhizopus oryzae aspartic protease, Pythium spp. cellulase, and Pythium spp. lipase. Useful signal peptides for yeast host cells include, but are not limited to, those derived from Saccharomyces cerevisiae α-factor andThose genes of Saccharomyces cerevisiae invertase. Useful signal peptides for mammalian host cells include, but are not limited to, those from genes of immunoglobulin γ (IgG). In some embodiments, the signal peptide comprises a naturally occurring signal sequence in human acid α-glucosidase. In some embodiments, the encoded signal peptide comprises a sequence containing residues 1 to 19 of SEQ ID NO: 2 or 12.

[0247] In some embodiments, the control sequence comprises one or more regulatory sequences that facilitate the regulation of the expression of polynucleotides and / or corresponding encoded polypeptides relative to the growth of the host cell. Examples of regulatory systems are those that cause gene expression to be turned on or off in response to chemical or physical stimuli, including the presence of regulatory compounds, as described on page 56 of this specification 52 / 115, CN 121511299 A. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to, the lactose operon, the tac operon, and the tryptophan operon system. 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 α-amylase promoter, the Aspergillus niger glucosylase promoter, and the Aspergillus oryzae glucosylase promoter. Exemplary inducible promoters regulated by exogenous factors include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible promoter, the mouse mammary tumor virus (MMTV) promoter; the ecdysone insect promoter, the tetracycline inducible promoter system, the RU486 inducible promoter system, and the rapamycin inducible promoter system.

[0248] In some embodiments, the recombinant expression vector can be any suitable vector (e.g., plasmid or virus, including but not limited to adenovirus (AV), adeno-associated virus (AAV), lentivirus (LV), and nonviral vectors such as liposomes and exosomes) that can readily undergo recombinant DNA procedures and can induce the expression of engineered acidic α-glucosidase polynucleotide sequences. The choice of vector will generally depend on the compatibility of the vector with the host cell to which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0249] In some embodiments, the expression vector is a self-replicating vector (i.e., a vector existing as an extrachromosomal entity whose replication process is independent of chromosome replication, such as plasmids, extrachromosomal elements, microchromosomes, or artificial chromosomes). The vector may contain any means to ensure self-replication. In some alternative embodiments, the vector may be a vector that integrates into the genome when introduced into a host cell and replicates along with one or more chromosomes into which it has been integrated. In some embodiments, the vector is a non-replicating and non-integrating vector that may exist in free form. Furthermore, a single vector or plasmid, or two or more vectors or plasmids (which together contain the total genome to be introduced into the host cell genome) may be used.DNA), or transposons may be used.

[0250] In some embodiments, recombinant polynucleotides may be provided on a non-replicating expression vector or plasmid. In some embodiments, the non-replicating expression vector or plasmid may be based on a replication-defective viral vector (see, for example, Travieso et al., npj Vaccines, 2022, Vol. 7, Article 75).

[0251] In some embodiments, the expression vector contains one or more selective markers that enable easy selection of transformed cells. “Selective marker” is a gene whose product provides resistance to biocides or viruses, resistance to heavy metals, from prototrophic to auxotrophic, etc. Examples of bacterial selective markers include, but are not limited to, the dal gene from Bacillus subtilis or Bacillus licheniformis, or markers that confer resistance to antibiotics (such as ampicillin, kanamycin, chloramphenicol, or tetracycline). Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selective markers for use in filamentous fungal host cells include, but are not limited to: amdS (acetamidinase; e.g., from Aspergillus nidulans or Aspergillus oryzae), argB (ornithine carbamoyltransferase), bar (phosphinicotinamide acetyltransferase; e.g., from Streptomyces hygromycin), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase; e.g., from Aspergillus nidulans or Aspergillus oryzae), sC (adenosyl sulfate transferase), and trpC (o-aminobenzoic acid synthase), and their equivalents. Selective markers for mammalian cells include, but are not limited to: chloramphenicol acetyltransferase (CAT), noromycin N-acetyltransferase, blastcin-S deaminase, blastcin-S acetyltransferase, Shble (Zeocin® resistance), aminoglycoside 3'-phosphotransferase (neomycin resistance), hph (hygromycin resistance), thymidine kinase, and puromycin N-acetyltransferase.

[0252] In another aspect, this disclosure provides a host cell comprising a recombinant polynucleotide encoding an engineered acidic α-glucosidase polypeptide described herein, the polynucleotide being operatively linked to one or more control sequences for the expression of one or more engineered acidic α-glucosidase polypeptides in the host cell. In some embodiments, the host cell is a prokaryotic or eukaryotic cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human cell. In some embodiments, the host cell is a cell lacking acidic α-glucosidase activity, or a cell obtained from a subject suffering from Pompe disease.

[0253] The host cells used for expressing the polypeptide encoded by the expression vector of this disclosure are known in the art and include, but are not limited to: fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris (e.g., ATCC Registry No. 201178)); insect cells (e.g., Drosophila S2 cells and Noctua sf9 cells), plant cells, animal cells (e.g., CHO, CHO-K1, COS, and BHK cells), and human cells (e.g., HEK293T cells, human fibroblasts, THP-1 cells, Jurkat cells, and Bowes melanoma cell lines). In some embodiments, the host cells are cells obtained from mammals, including model animals lacking acid α-glucosidase activity or human patients. In some embodiments, the host cells used for expression are cells obtained from human patients diagnosed with Pompe disease.

[0254] In another aspect, this disclosure provides a method for producing engineered acidic α-glucosidase peptides, wherein the method comprises culturing host cells capable of expressing polynucleotides encoding engineered acidic α-glucosidase peptides under conditions suitable for the expression of engineered acidic α-glucosidase peptides, thereby producing engineered acidic α-glucosidase peptides. In some embodiments, the method further comprises steps such as isolating the engineered acidic α-glucosidase peptides from culture media and / or cells. In some embodiments, the method further comprises purifying the expressed engineered acidic α-glucosidase, as described herein.

[0255] Suitable culture media and growth conditions for host cells described above are known in the art. The polynucleotides expressing engineered acidic α-glucosidase peptides can be introduced into cells by a variety of methods known in the art. Techniques include, but are not limited to, electroporation, bioprojectile particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0256] In some embodiments, engineered acidic α-glucosidase peptides having the properties disclosed herein can be obtained by subjecting a polynucleotide encoding a naturally occurring or engineered acidic α-glucosidase peptide to mutagenesis and / or directed evolution methods known in the art and as described herein. Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling (see, for example, Stemmer, Proc. Natl. Acad. Sci. USA, 1994, 91:10747-10751; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767 and U.S. Patent 6,537,746). Other directed evolution procedures that may be used include, but are not limited to, the staggered extension method (StEP), in vitro recombination (see, for example, Zhao et al., Nat. Biotechnol., 1998,16:258–261), mutagenesis PCR (see, for example, Caldwell et al., PCR Methods Appl., 1994, 3: S136–S140) and cassette mutagenesis (see, for example, Black et al., Proc. Natl. Acad. Sci. USA, 1996, 93:3525–3529).

[0257] Mutagenesis and directed evolution methods can be applied to polynucleotides to generate variant libraries that can be expressed, screened, and measured. Any suitable mutagenesis and directed evolution methods can be used in this disclosure (see, for example, 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, 6,277,638, 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, 6,376,246, 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, 6,489,146, 6,506,602, 6,506,603, 6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467, 6,579, Instruction manual, pages 54 / 115, 58 CN 121511299 A 678, 6,586,182, 6,602,986, 6,613,514, 6,653,072, 6,716,631, 6,946,296, 6,961,664, 6,995,017、7,024,312、7,058,515、7,105,297、7,148,054、7,288,375、7,421 ,347、7,430, 477、7,534,564、7,620,500、7,620,502、7,629,170、7,702,464、7,747,391、7,747,393、7, 751 ,986、7,776,598、7,783,428、7,795,030、7,853,410、7,868,138、7,873,499、7,904, 249, 7,957,912, 8,383,346, 8,504,498, 8,849,575, 8,876,066, 8,768,871, 9,593,326 and all related non-US corresponding patent applications; Ling et al., Anal. Biochem., 1997, 254(2):157-78; Dale et al., Meth. Mol. Biol., 1996, 57:369-74; Smith, Ann. Rev. Genet., 1985, 19:423-462; Botstein et al., Science, 1985, 229:1193-1201; Carter, Biochem. J., 1986, 237: 1-7; Kramer et al., Cell, 1984, 38:879-887; Wells et al., Gene, 1985, 34:315-323; Minshull et al., Curr. Op. Chem. Biol., 1999, 3:284-290; Christians et al., Nat. Biotechnol., 1999, 17:259-264; Crameri et al., Nature, 1998, 391:288-291; Crameri et al., Nat. Biotechnol., 1997, 15:436-438; Zhang et al., Proc. Nat. Acad. Sci. USA, 1997, 94:4504-4509; Crameri et al., Nat. Biotechnol., 1996, 14: 315‑319; Stemmer, Nature, 1994, 370:389‑391; Stemmer, Proc.Nat. Acad.Sci.USA, 1994,91:10747-10751; US ​​Patent Application Publications 2008 / 0220990, US 2009 / 0312196, US2014 / 0005057, US2014 / 0214391, US2014 / 0221216; US2015 / 0050658, US2015 / 0133307, US2015 / 0134315 and all related non-US corresponding patent applications; WO 95 / 22625, WO 97 / 0078, WO 97 / 35966, WO 98 / 27230, WO 00 / 42651, WO 01 / 75767 and WO 2009 / 152336; all of which are incorporated herein by reference.

[0258] In some embodiments, peptide variants obtained after mutagenesis are screened by subjecting the peptide variants to assay conditions (such as defined temperature, acidic or alkaline pH, serum / plasma exposure) and measuring the amount of residual peptide activity after treatment or other assay conditions. DNA containing the polynucleotide encoding the engineered acid α-glucosidase peptide is then isolated from host cells, sequenced to identify nucleotide sequence variations (if any), and used for protein expression in different or the same host cells. Activity from the expression library can be measured using any suitable method known in the art and as provided in the examples.

[0259] For engineered peptides with known sequences, the polynucleotide encoding the peptide can be prepared using standard solid-phase methods according to known synthetic methods. In some embodiments, polynucleotide fragments can be synthesized separately and then ligated (e.g., by enzymatic or chemical ligation methods, or polymerase-mediated methods) to form any desired continuous sequence. For example, the polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classic phosphoramidite method (see, for example, Beaucage et al., Tetra. Lett., 1981, 22:1859-69; and Mattes et al., EMBO J., 1984, 3:801-05), as is typically practiced in automated synthetic methods. According to the phosphoramidite method, the oligonucleotides are synthesized (e.g., in an automated DNA synthesizer), purified, annealed, ligated, and cloned into a suitable vector.

[0260] Thus, in some embodiments, a method for preparing engineered acid α-glucosidase polypeptides may include: (a) synthesizing a polynucleotide encoding an engineered acid α-glucosidase, such as the amino acid sequence of any variant provided in Table 3-1, and SEQ ID NO. of even numbers in SEQ ID NO: 14 to 754; and (b) expressing the engineered acid α-glucosidase polypeptide encoded by the polynucleotide. In some embodiments, the amino acid sequence optionally has 1 to 2, 1 to 3 amino acids.One to four, one to five, one to six, one to seven, one to eight, one to nine, one to ten, one to fifteen, one to twenty, one to twenty-one, one to twenty-one, one to twenty-two, one to twenty-two, one to twenty-three, one to twenty-four, one to twenty-five, one to thirty, one to thirty-five, one to forty, one to forty-five, or one to fifty amino acid residues are deleted, inserted, or substituted. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residues deleted, inserted, and / or substituted. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residues deleted, inserted, and / or substituted. In some embodiments, the substitution is a conserved substitution or a non-conserved substitution.

[0261] Any desired improved properties (e.g., activity / efficacy, stability, serum / plasma stability, alkaline pH tolerance, acidic pH tolerance, cellular uptake, etc.) of the expressed engineered acid α-glucosidase polypeptide can be evaluated using any suitable assays known in the art (including, but not limited to, the assays and conditions described herein).

[0262] Compositions

[0263] In another aspect, this disclosure provides compositions comprising an engineered acid α-glucosidase or a recombinant polynucleotide encoding such acid α-glucosidase, including, but not limited to, those compositions described below. In some embodiments, the composition comprises at least the engineered acidic α-glucosidase or recombinant polynucleotide exemplified in Tables 3-1 and 4-1 and the sequence listing.

[0264] Depending on the composition and method of administration, compositions comprising the engineered acidic α-glucosidase described herein are in solid, semi-solid, or liquid form. In some embodiments, the composition comprises other pharmaceutically acceptable ingredients, such as diluents, buffers, excipients, salts, emulsifiers, preservatives, stabilizers, fillers, and other ingredients. In some embodiments, compositions comprising the engineered acidic α-glucosidase polypeptide of the present disclosure comprise one or more commonly used carrier compounds, including but not limited to sugars (e.g., lactose, sucrose, mannitol, and / or sorbitol), starch, cellulose (e.g., methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose), gums (e.g., gum arabic, tragacanth gum, etc.).Guar gum, etc.) and / or proteins (e.g., gelatin, collagen, etc.). Detailed information on techniques for formulation and application is available in the art and described in the literature.

[0265] In some embodiments, the engineered acidic α-glucosidase peptide is formulated for use in a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient. Any suitable form for use in the delivery of the engineered acidic α-glucosidase peptide may be used herein, including but not limited to pills, tablets, gel tablets, capsules, lozenges, sugar-coated pills, powders, soft capsules, sol-gels, gels, emulsions, implants, patches, sprays, ointments, liniments, creams, pastes, gels, smears, aerosols, chewing gum, lubricants, sticks, solutions, suspensions (including but not limited to oil-based suspensions, oil-in-water emulsions, etc.), slurries, syrups, controlled-release agents, etc. In some embodiments, the engineered acidic α-glucosidase polypeptide is provided in a form suitable for injection or infusion (i.e., in injectable formulation), particularly for parenteral administration or infusion to patients (particularly human patients).

[0266] In some embodiments, the engineered acidic α-glucosidase polypeptide is provided in a biocompatible matrix (such as a sol-gel, including silica-based (e.g., oxysilane) sol-gels). In some embodiments, the engineered acidic α-glucosidase polypeptide is encapsulated. In some alternative embodiments, the engineered acidic α-glucosidase polypeptide is encapsulated in a nanostructure (e.g., nanotubes, nanochannels, nanocapsules or microcapsules, microspheres, liposomes, etc.). The engineered acidic α-glucosidase polypeptide is intended to be administered by any suitable means known in the art (including but not limited to parenteral administration (e.g., intravenous, intramuscular, subcutaneous, etc.), oral, topical, transdermal, intranasal, intraocular, intrathecal, via implant, etc.).

[0267] In some embodiments, the engineered acidic α-glucosidase polypeptide is chemically modified by glycosylation, chemical cross-linking agents, polyethylene glycolation (i.e., modification with polyethylene glycol [PEG] or activated PEG, etc.) or other compounds (see, for example, Ikeda, Amino Acids, 2005, 29:283-287; U.S. Patent Nos. 7,531,341, 7,534,595 and 7,560,263; U.S. Patent Publications Nos. 2013 / 0039898, 2012 / 0177722, etc.).

[0268] In some other embodiments, the engineered acidic α-glucosidase polypeptide is provided in the form of a formulation comprising matrix-stabilized enzyme crystals, as specified in the specification on page 56 / 115 of CN 121511299 A. In some embodiments, the formulation comprises cross-linked crystalline engineered acidic α-glucosidase.Enzymes and polymers having reactive portions that adhere to enzyme crystals. The present invention also provides engineered acidic α-glucosidase polypeptides in polymers.

[0269] In some embodiments, compositions comprising engineered acidic α-glucosidase polypeptides include one or more commonly used carrier compounds, including but not limited to sugars (e.g., lactose, sucrose, mannitol and / or sorbitol), starches (e.g., corn starch, wheat starch, rice starch, potato starch or other plant starches), celluloses (e.g., methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose), gums (e.g., gum arabic, tragacanth, guar gum, etc.) and / or proteins (e.g., gelatin, collagen, etc.). Other components in the oral formulation may include colorants and / or sweeteners (e.g., glucose, sucrose and mannitol) and lubricants (e.g., magnesium stearate) and enteric coatings (e.g., methacrylate polymers, hydroxypropyl methylcellulose phthalates and / or any other suitable enteric coating known in the art). In some embodiments, a disintegrant or solubilizer (e.g., cross-linked polyvinylpyrrolidone, agar, alginate, or salts thereof such as sodium alginate) is included. In some embodiments, the engineered acidic α-glucosidase peptide is combined with a variety of other components, including but not limited to preservatives, suspending agents, thickeners, wetting agents, alcohols, fatty acids, and / or emulsifiers, particularly in liquid formulations. In some embodiments, the engineered acidic α-glucosidase peptide is administered to a subject in combination with other compounds, molecules, and / or materials (including but not limited to pharmacological companions) for the treatment of Pompe disease and any other suitable compounds.

[0270] In some embodiments, the pharmaceutical composition comprises a recombinant polynucleotide encoding the engineered acidic α-glucosidase described herein. In some embodiments, the recombinant polynucleotide is DNA or mRNA. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient suitable for administration of the recombinant polynucleotide. In some embodiments, pharmaceutical compositions comprising a recombinant polynucleotide encoding an engineered acidic α-glucosidase are suitable for any suitable mode of administration, including but not limited to intravenous, intramuscular, subcutaneous, oral, intranasal, intraocular, intrathecal, inhalation, etc.

[0271] In some other embodiments, a recombinant polynucleotide encoding an engineered acidic α-glucosidase polypeptide is provided for delivery to cells or tissues via gene therapy, which includes viral delivery vectors (including but not limited to adenovirus (AV), adeno-associated virus (AAV), and lentivirus (LV)). In some embodiments, a recombinant polynucleotide encoding an engineered acidic α-glucosidase is provided for delivery in a non-viral vector or formulation (including but not limited to liposomes, nanotubes, nanochannels, nanocapsules, or microcapsules and microspheres). In some embodiments, an engineered acidic α-glucosidase is provided.Recombinant polynucleotides of enzyme peptides are used to formulate polynucleotide sequences in encapsulated delivery systems (such as liposomes) or to formulate lipid nanoparticles for delivery to cells or tissues via mRNA therapy (see, for example, Hou et al., Nature Reviews Materials, 2021, 6:1078–1094).

[0272] In some other embodiments, engineered acidic α-glucosidase peptides are provided for delivery to cells or tissues via cell therapy, wherein a polynucleotide sequence encoding the engineered acidic α-glucosidase peptide is introduced into a foreign cell, and the cell (or cells) is introduced into a recipient (e.g., a patient with Pompe disease or at risk of developing Pompe disease). Exemplary cells that may be used include, but are not limited to, hematopoietic (blood-forming) stem cells (HSCs), skeletal muscle stem cells, and mesenchymal stem cells.

[0273] Uses and Methods

[0274] In another aspect, this disclosure provides the use of engineered acid α-glucosidase peptides, recombinant polynucleotides encoding engineered acid α-glucosidase, or combinations thereof for treating a subject suffering from acid α-glucosidase activity deficiency. In some embodiments, the engineered acid α-glucosidase peptides, recombinant polynucleotides, or combinations thereof are used to treat one or more symptoms associated with acid α-glucosidase activity deficiency. In some embodiments, engineered acid α-glucosidase peptides, recombinant polynucleotides, or combinations thereof are used to treat a subject suffering from Pompe disease.

[0275] In some embodiments, a method for treating and / or preventing symptoms of acid α-glucosidase activity deficiency includes providing an effective amount of the engineered acid α-glucosidase described herein to a subject in need. In some embodiments, an effective amount of the engineered acid α-glucosidase or a pharmaceutical composition thereof is administered to the subject. In some embodiments, the engineered acidic α-glucosidase is administered at a dose of about 1 mg / kg to about 100 mg / kg. In some embodiments, the engineered acidic α-glucosidase is administered at a dose of about 10 mg / kg to about 60 mg / kg. In some embodiments, the engineered acidic α-glucosidase is administered at a dose of about 20 mg / kg to about 40 mg / kg.

[0276] In some embodiments, a recombinant polynucleotide encoding an engineered acidic α-glucosidase or a pharmaceutical composition thereof is administered to a subject. In some embodiments, a recombinant polynucleotide encoding an engineered acidic α-glucosidase or a pharmaceutical composition thereof is administered at a therapeutically effective dose. In some embodiments, the recombinant polynucleotide encoding an engineered acidic α-glucosidase or a pharmaceutical composition thereof is administered intravenously, intramuscularly, subcutaneously, intranasally, intraocularly, intrathecally, by inhalation, or orally.

[0277] In some embodiments, the subject receiving treatment suffers from Pompe disease. In some embodiments, the symptoms of Pompe disease improve. In some embodiments, the subject receiving treatment is an infant or child. In some embodiments, the subject receiving treatment is an adult or adolescent.

[0278] In some embodiments, this disclosure also provides the use of engineered acid α-glucosidase in the preparation of a medicament for treating acid α-glucosidase deficiency in a subject. In some embodiments, this disclosure provides the use of a recombinant polynucleotide encoding an engineered acid α-glucosidase in the preparation of a medicament for treating acid α-glucosidase activity deficiency in a subject. In some embodiments, the condition involving acid α-glucosidase deficiency is Pompe disease.

[0279] The foregoing and other aspects of the invention can be better understood in conjunction 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.

[0280] Examples

[0281] In the following experimental disclosures, the following abbreviations are used: ppm (parts per million); M (moles); mM (millimoles), μM and µM (micromoles); nM (nanomoles); mol (moles); gm and g (grams); mg (milligrams); ug and µg (micrograms); L and l (liters); ml and mL (milliliters); ul, μl, uL, μL (microliters); cm (centimeters); mm (millimeters); μm and µm (micrometers); sec. (seconds); min (minutes); h and hr (hours); U (units); MW (molecular weight); rpm (revolutions per minute); ℃ (degrees Celsius); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); Escherichia coli W3110 (a commonly used laboratory strain of Escherichia coli, available from the Coli Genetic Stock Center (CGSC) in New Haven, Connecticut, USA); DPBS (Döbek's phosphate buffer); LB (Luria-Burtani); TB (super broth); 4-MUGlu or 4-MU-GLU (4-methylumbelliferyl ketone α-D-glucopyranoside); SD-Ura (uracil-free single-deficient medium); HPLC (high-performance liquid chromatography); SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis); MU-Glu (4-methylumbelliferyl ketone α-D-glucopyranoside); IPTG (isopropyl β-D-1-thiogalactoside); PMBS (polymyxin B sulfate); FIOPC (fold improvement relative to positive control); PBMC (peripheral blood mononuclear cells); LB (Luria broth); and MeOH (methanol).

[0282] Example 1

[0283] GAA gene acquisition and expression vector construction

[0284] This example describes the acquisition of the GAA gene and the construction of the expression vector. A synthetic gene encoding WT human GAA (Uniprot ID P10253) was designed to optimize its gene expression in Homo sapiens (SEQ ID NO: 1) and cloned into the expression vector pDH (see International Patent Publication WO2021127457). For secretory expression and transient transfection in mammalian cells, a chimeric GAA expression construct encoding a synthetic mouse IG signal peptide (residues 1 to 19 of Uniprot accession number A0N1R5) was generated and fused with a synthetic gene encoding a different GAA variant, as described below. Oligonucleotides containing restriction endonuclease flanking sites that can be cloned into the BamHI / XhoI or HindIII / XhoI sites were used to amplify the fragment encoding the synthetic mouse IG signal peptide and the coding sequence for the mature form of GAA. Acidic α-glucosidase variants SEQ ID NO: 3, 7, 5, 13, 9 were cloned into pDH or pcDNA3.1(+). Using directed evolution, specific gene variants derived from SEQ ID NO: 11 (the polynucleotide sequence disclosed in WO2021127457 is SEQ ID NO: 3103, and the polypeptide sequence is SEQ ID NO: 3104) were generated in the pDH plasmid construct (see, for example, U.S. Patent Nos. 8,383,346 and WO2010 / 144103).

[0285] Example 2

[0286] High-throughput growth of suspension mammalian cells and assay of GAA obtained by suspension mammalian expression

[0287] High-throughput (HTP) growth of GAA and GAA variants in suspension mammalian cells (Expi293F)

[0288] EXPI293F cells (ThermoFisher Scientific) were transfected with polynucleotides encoding wild-type GAA or GAA variants having a synthetic mouse IG signal peptide fusion using liposome transfection 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 x 10⁶ cells / well / 400 µL in Axygen 1.1 mL deep-well plates (Corning, P-DW-11-C-S). Cells underwent liposome-mediated transfection and were returned to a culture medium with 8%...In a shaking incubator with CO2 and 70% humidity for 3 to 4 days, the GAA variant was expressed and secreted into the conditioned medium. The conditioned medium was harvested by centrifuging the expression plate and transferred to a BioRad Hardshell PCR plate (BioRad, HSP9601). The plate was centrifuged again and the clarified conditioned medium was transferred to a new 96-well plate for activity, stability, or cell uptake analysis.

[0289] HTP analysis of the supernatant

[0290] The activity of the GAA variant was determined by measuring the hydrolysis of 4-methylumbelliferyl ketone α-D-glucopyranoside (4-MU-GLU). For the unchallenged assay, 5 µL of clear EXPI293F™ conditioned medium prepared as described above was mixed with 50 µL of McIlvaine buffer (McIlvaine, J. Biol. Chem., 1921, 49:183-186) (pH 4.4) containing 1.5 mM 4-MU-GLU in a 96-well black opaque substrate. The reaction was incubated at 25 °C with stirring at 400 rpm for 15 min, and then quenched with 100 μL of 0.5 M sodium carbonate (pH 10.5). Fluorescence (Ex. 355 nm, emission 460 nm) was monitored using an EnVision microplate reader (Perkin Elmer) to analyze the hydrolysis. The unstimulated activity FIOPC was calculated by dividing the normalized GAA variant by the activity of the reference peptide with the indicated SEQ ID NO.

[0291] HTP Analysis of Plasma-Stimulated Supernatant

[0292] GAA variants were stimulated with plasma to simulate the conditions these variants encounter in the blood after administration to patients. First, 30 µL of EXPI293F™ clarified conditioned medium containing the GAA variants was combined with 30 µL of plasma (Innovative Research, Innovative Grade US Origin, cynomolgus monkey plasma K2 EDTA) in a 96-well plate. The plate was sealed and incubated at 37°C with stirring at 400 rpm for 4 h. Next, 10 µL of the plasma-stimulated sample was mixed with 50 µL of McIlvaine buffer (pH 4.4) containing 1.5 mM 4-MU-GLU. The reaction was incubated at 25°C to 37°C with stirring at 400 rpm for 30 min, and then quenched with 100 μL of 0.5 M sodium carbonate (pH 10.5). The hydrolysis was analyzed by monitoring fluorescence (Ex. 355 nm, emission 460 nm) using an EnVision microplate reader (Perkin Elmer).Plasma stability FIOPC was calculated by dividing the normalized GAA variant activity after stimulation by the activity of the reference peptide with the indicated SEQ ID NO after stimulation.

[0293] HTP analysis of GAA activity in Pompeii fibroblast lysates

[0294] GAA variants expressed by HTP EXPI293F™ from clarified conditioned medium were incubated with target cells, and residual intracellular activity was measured after 72 hours. For these experiments, mammalian cells lacking functional GAA activity, i.e., fibroblasts derived from Pompeii patients (Coriell Institute for Medical Research, #GM00248), were used. Pompeii patient-derived fibroblasts were seeded in 96-well COSTAR® (3904, Corning) plates and grown to confluence in standard complete growth medium. After confluence, the complete growth medium was removed from the plates using an automated BioMek i5 liquid processor. Clarified conditioned medium from transient HPT transfection in EXPI293F™ was transferred to fibroblasts derived from Pompe disease patients and incubated at 37°C and 5% CO2 for 24 hours. The medium was removed from the culture using an automated BioMek i5 liquid processor. Cells were simply washed with 150 μL of 1xDPBS per well, and the DPBS was removed using the automated BioMek i5 liquid processor. Then, 200 μL of standard complete growth medium was added to each well, and the plate was returned to the incubator for 72 hours. At the end of the incubation, the standard complete growth medium was removed using an automated BioMek i5 liquid processor. Cells were washed with 150 μL of 1xDPBS per well, and the DPBS was removed using an automated BioMek i5 liquid processor. Cell lysis was performed by adding 50 μL of McIlvaine buffer (pH 4.4) supplemented with 0.5% TRITON X-100™ nonionic surfactant (Sigma, catalog number 93443) and stirring at room temperature for 30 min. Activity was assessed by adding 50 µL of McIlvaine buffer (pH 4.4) containing 1.5 mM 4-MU-GLU. The plates were sealed and incubated at 37°C with stirring at 400 rpm for 360 min, followed by quenching with 100 μL of 0.5 M sodium carbonate (pH 10.5). Hydrolysis was analyzed using an EnVision microplate reader (Perkin Elmer) to monitor fluorescence (Ex. 355 nm, emission 460 nm). Intracellular activity of the normalized GAA variant was determined by dividing by the indicated SEQ ID.The activity of the reference peptide of NO was used to calculate cellular uptake FIOPC.

[0295] Example 3

[0296] GAA variant of SEQ ID NO: 11

[0297] In this example, experiments on the evolution and screening of a GAA variant derived from SEQ ID NO: 11 are described to obtain improved GAA activity after a series of stimuli. A library of variant genes encoded by SEQ ID NO: 11 was constructed and plated, grown, and screened to evaluate GAA 4-MU-GLU activity (“active FIOPC without stimulation”) and activity after plasma stimulation (“plasma stability and active FIOPC”), as described in Example 2. The 4-MU-GLU activity of the variant (“active FIOPC of lysates from Pompeii fibroblast treatment”) was also tested after treatment with conditioned medium on lysed Pompeii fibroblasts, as described in Example 2. The results of these assays are shown in Table 3-1. Instruction manual 60 / 115 pages 64 CN 121511299 A

[0298] Instruction manual 61 / 115 pages 65 CN 121511299 A Instruction manual 62 / 115 pages 66 CN 121511299 A Instruction manual 63 / 115 pages 67 CN 121511299 A Instruction manual 64 / 115 pages 68 CN 121511299 A Instruction manual 65 / 115 pages 69 CN 121511299 A Instruction manual 66 / 115 pages 70 CN 121511299 A Instruction manual 67 / 115 pages 71 CN 121511299 A Instruction manual 68 / 115 pages 72 CN 121511299 A Instruction manual 69 / 115 pages 73 CN 121511299 A Instruction manual 70 / 115 Page 74 CN 121511299 A Instruction Manual 71 / 115 Page 75 CN 121511299 A Instruction Manual 72 / 115 Page 76 CN 121511299 A Instruction Manual 73 / 115 Page 77 CN 121511299 A Instruction Manual 74 / 115 Page 78 CN 121511299 A Instruction Manual 75 / 115 Page 79 CN 121511299 A Instruction Manual 76 / 115 Page 80 CN 121511299 A Instruction Manual 77 / 115 Page 81 CN 121511299 A Instruction Manual 78 / 115 Page 82 CN 121511299 A Instruction Manual 79 / 115 Page 83 CN 121511299Instruction Manual A, page 80 / 115, 84 CN 121511299; Instruction Manual A, page 81 / 115, 85 CN 121511299; Instruction Manual A, page 82 / 115, 86 CN 121511299; Instruction Manual A, page 83 / 115, 87 CN 121511299; Instruction Manual A, page 84 / 115, 88 CN 121511299; Instruction Manual A, page 85 / 115, 89 CN 121511299; Instruction Manual A, page 86 / 115, 90 CN 121511299; Instruction Manual A, page 87 / 115, 91 CN 121511299; Instruction Manual A, page 88 / 115, 92 CN 121511299; Instruction Manual A, page 89 / 115, 93 CN 121511299; Instruction Manual A, page 90 / 115, 94 CN 121511299 A Instruction Manual 91 / 115 pages 95 CN 121511299 A Instruction Manual 92 / 115 pages 96 CN 121511299 A Instruction Manual 93 / 115 pages 97 CN 121511299 A Instruction Manual 94 / 115 pages 98 CN 121511299 A Instruction Manual 95 / 115 pages 99 CN 121511299 A Instruction Manual 96 / 115 pages 100 CN 121511299 A Instruction Manual 97 / 115 pages 101 CN 121511299 A Instruction Manual 98 / 115 pages 102 CN 121511299 A Instruction Manual 99 / 115 pages 103 CN 121511299 A Instruction Manual 100 / 115 pages 104 CN 121511299 A Instruction manual 101 / 115 pages 105 CN 121511299 A

[0299] Example 4

[0300] Production of GAA variants

[0301] Production of GAA in EXPI293F™ cells

[0302] Using EXPIFECTAMINE™ 293 reagent (ThermoFisher Scientific) in EXPI293™ expression medium (ThermoFisher Scientific), EXPI293F™ cells (ThermoFisher Scientific) were transiently transfected via liposome transfection to achieve milligram-level production of GAA variants. The GAA variant fused to the N-terminus to synthesize a mammalian signal peptide was subcloned into the mammalian expression vector pDH or pcDNA 3In .1(+), as described in Example 1. EXPI293F™ cells were transfected with plasmid DNA and grown in suspension for 4 to 7 days. The conditioned medium was then harvested, clarified by centrifugation and filtration or diatomaceous earth, and stored at -80°C until purification.

[0303] Example 5

[0304] Purification of GAA variants

[0305] GAA variants (SEQ ID NO: 2, 4, 12, 8, 6, 14, 10) generated in EXPI293F™ cells as described in Example 4 were purified from mammalian culture supernatant as described in the literature (Yasuda et al., Prot. Exp. Pur., 2004, 37:499-506). Concanavalin A resin (Sigma Aldrich) was equilibrated with 0.1 M sodium acetate, 0.1 M NaCl, 1 mM MgCl2, CaCl2, and MnCl2 (pH 6.0) (concanavalin A binding buffer). The supernatant was concentrated 10-fold and diluted 2-fold with equilibration buffer (0.1 M sodium acetate, 0.1 M NaCl, 1 mM MgCl2, CaCl2, and MnCl2, pH 6.0) before being loaded onto a 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 the buffer was exchanged for 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 filter unit (Millipore) with a 50 kDa molecular weight cutoff membrane. The GAA in the storage buffer was aseptically filtered through an ANOTOP® 0.2 µm syringe filter (Whatman) and stored at -80°C. The purification process produced 60 to 100 mg of purified protein per liter of culture supernatant.

[0306] Example 6

[0307] In vitro characterization of GAA variants 102 / 115 pages 106 CN 121511299 A

[0308] In this example, experiments performed to characterize the GAA variants produced as described herein are described.

[0309] Stability of rhGAA and GAA variants at neutral pH

[0310] The stability of the variant at neutral pH was determined by incubating the purified GAA variant at 214 nM in MEM complete growth medium (pH 7.4) in 96-well plates. The plates were incubated at 37°C for up to 144 hours. At each time point, 10µL of sample stimulated at neutral pH was transferred to BioRad hardshell plates and immediately frozen at -80°C. After the experiment, all plates were simultaneously treated with 50 μL of McIlvaine buffer containing 1.5 mM 4-MU-GLU for 30 minutes, with stirring at 400 rpm at 37°C. The reaction was quenched with 100 μL of Na2CO3 (0.5 M, pH 10.5), and 100 μL was transferred to black 96-well plates. Hydrolysis was assessed by quantifying the released fluorescent methylumbelliferone using an Envision microplate reader (ex 355 / em 460 nm). The results from this assay are shown in Figure 1.

[0311] Hemolysis temperatures of rhGAA and GAA variants at neutral and lysosomal pH

[0312] The hemolysis temperatures of rhGAA and GAA variants at neutral and lysosomal pH were determined by differential scanning fluorometry. The GAA enzyme variants were diluted to 1 mg / mL with DPBS (pH 6.2). 40 µL of pH 4.4 or pH 7.4 McIlvaine buffer containing 1X SYPRO Orange was added to 10 µL of each enzyme solution (n = 3) in individual wells of a 96-well BioRad skirted plate. The plate was sealed with an optically clear membrane and run at 25°C to 95°C using the manufacturer's recommended method on a CFX Connect real-time quantitative PCR system. Data were analyzed using BioRad software. The results of these assays are shown in Figure 2.

[0313] Stability of rhGAA and GAA variants in plasma

[0314] The stability of the GAA variants in cynomolgus monkey plasma was assessed over a 75-hour timeframe. The purified GAA variant was diluted in GAA storage buffer (20 mM sodium phosphate, 150 mM NaCl, 185 μM polysorbate 20, pH 6.0) to a concentration of 100 μg / mL (2x assay concentration) and aliquoted into BioRad hardshell plates in triplicate (100 µL / well). 100 µL of cynomolgus monkey plasma was added and thoroughly mixed. The plasma stimulation plates were sealed and incubated at 37°C with stirring (400 rpm) for up to 75 hours. At various time points during stimulation, the residual activity of the GAA variant was measured by transferring 10 μL of the plasma stimulation solution to 50 μL of McIlvaine buffer (pH 4.4) containing 1.5 mM 4-MU-GLU in black 96-well plates for 30 minutes with stirring at 37°C (400 rpm). The reaction was quenched with 100 μL Na₂CO₃ (0.5 M, pH 10.5) and analyzed using an Envision microplate reader (ex 355 / em 460).The released fluorescent methylumbelliferone was quantified (nm) to assess hydrolysis. Results from this assay are shown in Figure 3.

[0315] Cellular uptake of purified GAA variants in Pompe Fibroblasts or C2C12 GAA knockout myoblasts

[0316] The ability of the GAA variants to cross-correct cells compared to reference enzymes (SEQ ID NO: 2 and 4) was determined. Pompe Fibroblasts (GM00248, Coriell Institute for Medical Research) or C2C12 GAA knockout myoblasts were seeded in standard complete growth medium in black-walled, clear-bottomed 96-well plates (Costar, #3604) and allowed to reach confluence (2 to 3 days at 37°C, 5% CO2). After reaching confluence, the standard complete growth medium was removed using an automated BioMek i5 liquid processor. The purified enzyme, as described in Example 5, was added to cells in serial dilutions ranging from 0 to 214 nM GAA / mL in standard complete growth medium and incubated at 37°C and 5% CO2 for 1, 4, 24, or 96 hours. After treatment, the medium containing the GAA variant was aspirated using an automated BioMek i5 liquid processor. Cells were simply washed with 150 μL of 1xDPBS per well, and the DPBS was removed using the automated BioMek i5 liquid processor. Then, 200 μL of standard complete growth medium was added to each well, and the plate was returned to the incubator for the remaining 96 hours of experiment (the duration could be 95 hours to 0 hours depending on the treatment time). At the end of the experiment, the MEM complete growth medium was removed using an automated BioMek i5 liquid processor. Cells were washed with 150 μL of 1xDPBS per well, and the DPBS was removed using the automated BioMek i5 liquid processor. Cells were lysed by adding 50 μL of McIlvaine buffer (pH 4.4) supplemented with 0.5% TRITON X-100™ nonionic surfactant (Sigma #93443) and stirring at room temperature for 30 minutes. GAA activity was assessed by adding 50 µL of McIlvaine buffer (pH 4.4) containing 1.5 mM 4-MU-GLU. The plates were sealed and incubated at 37°C with stirring at 400 rpm for 300 to 360 minutes, followed by quenching with 100 μL of 0.5 M sodium carbonate (pH 10.5). Fluorescence (Ex. 355 nm, emission 460 nm) was monitored using an EnVision (Perkin Elmer) microplate reader.Hydrolysis was analyzed. Figures A, B, C, and D in Figure 4 provide graphs showing the activity of Pompeii fibroblast lysates after treatment with purified GAA variants for 1 to 96 hours. Figures A, B, C, and D in Figure 5 provide graphs showing the activity of purified GAA variants in C2C12 GAA KO myoblast lysates (1 to 96 hours).

[0317] Example 7

[0318] In vitro characterization of GAA variant expression in myoblasts

[0319] In this example, experiments performed to characterize the expression and activity of GAA variants in myoblasts as described herein are described.

[0320] rhGAA and GAA variant 4-MU-GLU and glycogen hydrolysis activity in transiently transfected myoblasts

[0321] The expression efficiency and activity of wild-type GAA and GAA variants in myoblasts were assessed using transient transfection. C2C12 GAA knockout myoblasts were seeded in complete growth medium (Dürbeck modified Eagle medium containing 10% fetal bovine serum) in 12-well plates and allowed to adhere for 24 hours to achieve approximately 50% confluence. Cells were transfected with plasmid DNA of the GAA variant using jetOPTIMUS® Polyplus transfection reagent. The plates were returned to the incubator for 4 hours, followed by medium replacement. Three days post-transfection, GAA activity in the conditioned medium was assessed, and myoblasts were harvested to assess GAA activity in the lysates. GAA activity in the conditioned medium was determined relative to 4-MU-GLU and was assessed by incubating 20 μL of conditioned medium with 50 μL of McIlvaine buffer (pH 4.4) containing 1.5 mM 4-MU-GLU in black 96-well plates at 37°C with stirring (400 rpm) for 4 hours. Hydrolysis was assessed by quantifying the released fluorescent methylumbelliferone using an Envision microplate reader (ex 355 / em 460 nm). To generate lysates, myoblasts were harvested with trypsin and centrifuged. The cell pellet was washed with DPBS and lysed on ice with 52 μL of GAA lysis buffer (0.2 M sodium acetate, 0.4 M potassium chloride, 0.5% Triton X-100, pH 4.3) for 30 min with intermittent vortexing. The lysates were clarified (20,000 RCF, 10 min), and protein concentrations were determined by BCA and normalized. GAA activity relative to 4-MU-GLU in the lysates was assessed by incubating 2 μL or 4 μL of normalized lysates with 50 μL of McIlvaine buffer (pH 4.4) containing 1.5 mM 4-MU-GLU in black 96-well plates at 37°C with stirring (400 rpm) for 4 h. By using the Envision microplate reader (ex 355 / The released fluorescent methylumbelliferone was quantitatively analyzed at 460 nm (em) to assess hydrolysis. The glycogenolytic activity of the lysate was determined by incubating it with glycogen (100 mg / mL, prepared in GAA reaction buffer (pH 4.3) containing 0.1 M sodium acetate, 0.1 M NaCl, and 0.5 mg / mL BSA) at 37 °C and 400 rpm for 1 h. Glycogenolysis was quenched / neutralized by adding 90 μL of stop buffer (133 mM glycine, 83 mM sodium carbonate, pH 10.7). The quenched hydrolysis was then diluted 1:20 with Amplex Red reaction buffer (Invitrogen Amplex Red Glucose / Glucose Oxidase Assay Kit #A22189). Add 50 μL of quenched hydrolysis reaction dilution buffer and 50 μL of glucose standards (concentrations of 0, 3.2, 6.25, 12.5, 25, 50, and 100 μM) to black 96-well plates. Add 50 μL of Amplex reagent mixture (Amplex Red / HRP / glucose oxidase) to all wells, and incubate the plates at room temperature with gentle shaking in the dark for 30 minutes. Quantify the red fluorescent reagent halogen (formed by the reaction of Amplex Red with hydrogen peroxide generated from the coupling reaction of glucose oxidase–HRP) using a Spectramax EM microplate reader (ex 540 / em 590 nm). Figure 6 shows the activity of C2C12 GAA KO myoblasts in conditioned medium after transient transfection. Figure 7 shows (Figures A and B) the activity of C2C12 GAA KO myoblasts in lysates after transient transfection.

[0322] Example 8 Specification 104 / 115 pages 108 CN 121511299 A

[0323] Identification of active GAA variants with reduced immunogenicity

[0324] In this example, experiments performed to characterize the expression and activity of GAA variants in myoblasts as described herein are described.

[0325] The putative T-cell epitopes in SEQ ID NO: 2 of WT GAA or engineered GAA variant SEQ ID NO: 12 were identified using tools from the Immunotope Database (IEDB; Immunotope Database and Analysis Resource Website) (as known in the art) and proprietary statistical analysis tools (see, for example, iedb.org; and Vita et al., Nucl. Acids Res., 2020, 38 (Database Special Issue): D854-62. Electronic version November 11, 2009). WT GAA or engineered GAA variants were resolved into all possible 15-mer analytical frames, with each frame overlapping the previous frame by 14 amino acids.The immunogenic potential of these analytical frameworks was assessed by scoring the predicted combinations of the 15-mer analytical framework with eight common class II HLA-DR alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, and DRB1*1501) using methods recommended on the IEBDB website. These alleles collectively cover 77% of the world's population (see, for example, iedb.org; and Bui et al., 2006, BMC Bioinformatics, 7:153). Statistical analysis tools known in the art were used to identify potential T-cell epitope clusters contained within the enzyme (i.e., subregions within GAAs with exceptionally high immunogenic potential).

[0326] For the GAA variants identified in Example 3 that were active in the assay described in Example 2, their predicted immunogenicity levels were analyzed by assessing their binding to eight common class II HLA-DR alleles. A total immunogenicity score and immunogenicity hit count were calculated for each variant. The total immunogenicity score (TIS) reflects the total number of predicted binding events of the eight MHC class II alleles (as described above) with peptide 15-mer in the sequence (i.e., a higher score indicates a higher predicted immunogenicity level). The immunogenicity hit count (IHC) indicates the number of peptide 15-mers predicted to bind to four or more of the eight common alleles; these sequence regions have particularly high immunogenicity potential in the population (i.e., a higher score indicates a higher immunogenicity potential). Mutations that result in a decrease in the total immunogenicity score and / or immunogenicity hit count compared to the reference sequence were considered potential "deimmunizing mutations" and are shown in Table 4-1. Instruction manual, pages 105 / 115, 109 CN 121511299 A

[0327] ; Instruction manual, pages 106 / 115, 110 CN 121511299 A; Instruction manual, pages 107 / 115, 111 CN 121511299 A; Instruction manual, pages 108 / 115, 112 CN 121511299 A; Instruction manual, pages 109 / 115, 113 CN 121511299 A; Instruction manual, pages 110 / 115, 114 CN 121511299 A; Instruction manual, pages 111 / 115, 115 CN 121511299 A; Instruction manual, pages 112 / 115, 116 CN 121511299 A; Instruction manual, pages 113 / 115, 117 CN 121511299 A; Instruction manual, pages 114 / 115, 118 CN 121511299 A

[0328] Example 9

[0329] Characterization of GAA variants in in vitro immunogenicity assessment

[0330] MHC II-related peptide proteomics (MAPP) assays can be used to provide experimental evidence of HLA-binding epitopes from protein antigens. MAPP assays combine the main steps of antigen uptake into differentiated antigen-presenting cells, lysosomal processing, HLA binding, and presentation at the cell surface of antigen-presenting cells. Naturally processed, bound, and presented peptides are then identified and quantified by liquid chromatography-mass spectrometry. Presentation of peptides to CD4+ helper T cells at the cell surface by HLA-DR receptors is a necessary step in activating the cascade of reactions that include T cell proliferation, differentiation, and ultimately antibody production by B cells. Therefore, reduced binding of treated antigens to the grooves of MHC II molecules and subsequent reduced antigen presentation are considered to reduce the potential immunogenic risk of the antigen.

[0331] In this example, peripheral blood mononuclear cells (PBMCs) were isolated from the erythrocyte sedimentation rate (ESR) layer of healthy donors to isolate CD14+ mononuclear cells, which were then differentiated into dendritic cells (DCs) using methods known in the art. The DCs were then incubated with GAA variants and induced to acquire a mature phenotype by the addition of lipopolysaccharide. HLA-DR binding peptides from the processed antigens (GAA and its variants) were captured by immunoprecipitation and eluted for LC-MS analysis. The identified binding peptides were compared with the amino acid sequences of the GAA variants to compare the processing and abundance of peptides derived from each GAA variant (Kropshofer and Spindeldreher (2005), in Antigen Presenting Cells: From Mechanisms to Drug Development, eds. Kropshofer and Vogt, Wiley-VCH, Weinheim, 159-98). By comparing the identified peptides, experiments showed that the GAA variants of SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 14 had significantly reduced processing and lower peptide presentation frequency compared to SEQ ID NO: 2 (see Figure 8).

[0332] Although the invention has been described with reference to specific embodiments, various modifications and equivalents may be made to suit particular circumstances, materials, compositions, methods, or one or more method steps, thereby achieving the benefits of the invention without departing from the scope of the claims.

[0333] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated in their entirety for all purposes, as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated in its entirety for all purposes. Specification 115 / 115 pages 119 CNFigure 1. Instruction Manual Appendix 1 / 8 Page 120 CN 121511299 A Figure 2. Instruction Manual Appendix 2 / 8 Page 121 CN 121511299 A Figure 3. Instruction Manual Appendix 3 / 8 Page 122 CN 121511299 A Figure 4. Instruction Manual Appendix 4 / 8 Page 123 CN 121511299 A Figure 5. Instruction Manual Appendix 5 / 8 Page 124 CN 121511299 A Figure 6. Instruction Manual Appendix 6 / 8 Page 125 CN 121511299 A Figure 7. Instruction Manual Appendix 7 / 8 Page 126 CN 121511299 A Figure 8. Instruction Manual Appendix 8 / 8 Page 127 CN 121511299 A

Claims

1. An engineered acidic α-glucosidase or a bioactive fragment thereof, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 12, wherein said amino acid sequence comprises at least the substitutions or amino acid residues 305V, 24A / C / D / F / G / H / I / K / M / N / P / S / T / V / Y, 28A / C / D / E / F / G / H / K / Q / T / V / W, 29A / C / D / E / F / G / H / I / K / M / N / P / R / W / Y, 39A / E / F / G / I / L / N / T, 50A / C / D / E / F / H / I / K / M / N / R / S / T / W / Y, 62D / H / I / K / M / N / P / Q / Y, 78A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 87A / G / H / I / K / L / MN / Q / R / S / T / V / W, 135C / D / E / F / G / H / I / K / L / N / R / Y, 266A / D / E / H / K / Q, 267 H / L / T / V, 437A / H, 486C / D / F / G / H / I / K / L / M / N / Q / R / S / V / W / Y, 522A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y, 569A / C / D / E / G / K / M / N / P / R / W, 670A / D / G / H / K / M / Y, 692A / D / E / H / K / L / M / N / T / W, 711D / E / I / K / M / N / Q / S / T / V / Y, 736F / L, 750E / K / L / Q / R, 81 2A / D / G / S, 830D / E / F / G / H / L / M / N / S / T / W / Y, 842A / C / D / F / H / K / L / M / N / Q / R / T / W, 871A / C / D / F / H / I / M / N / Q / T / V / W / Y, 883A / F / Q, 894A / D / E / H / I / K / L / M / N / S / T / V / W / Y, 913F / I / K / M / N / S or 932C / D / E / G / H / K / L / M / N / P / Q / R / W / Y, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2, or relative to the reference sequence corresponding to SEQ ID NO: 12 or 2.

2. An engineered acidic α-glucosidase or a bioactive fragment thereof, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with the reference sequence of the even-numbered SEQ ID NOs in SEQ ID NOs: 14 to 754, wherein said amino acid sequence comprises at least the substitutions or amino acid residues 305V, 24A / C / D / F / G / H / I / K / M / N / P / S / T / V / Y, 28A / C / D / E / F / G / H / K / Q / T / V / W, 29A / C / D / E / F / G / H / I / K / M / N / P / R / W / Y, 39A / E / F / G / I / L / N / T, 50A / C / D / E / F / H / I / K / M / N / R / S / T / W / Y, 62D / H / I / K / M / N / P / Q / Y, 78A / C / D / F / G / H / I / K / L / M / N / Q / R / S / T / V / W / Y, 87A / G / H / I / K / L / MN / Q / R / S / T / V / W, 135C / D / E / F / G / H / I / K / L / N / R / Y, 266A / D / E / H / K / Q, 26 7H / L / T / V, 437A / H, 486C / D / F / G / H / I / K / L / M / N / Q / R / S / V / W / Y, 522A / C / D / F / G / H / I / K / L / M / N / P / Q / R / S / T / W / Y, 569A / C / D / E / G / K / M / N / P / R / W, 670A / D / G / H / K / M / Y, 692A / D / E / H / K / L / M / N / T / W, 711D / E / I / K / M / N / Q / S / T / V / Y, 736F / L, 750E / K / L / Q / R, 81 2A / D / G / S, 830D / E / F / G / H / L / M / N / S / T / W / Y, 842A / C / D / F / H / K / L / M / N / Q / R / T / W, 871A / C / D / F / H / I / M / N / Q / T / V / W / Y, 883A / F / Q, 894A / D / E / H / I / K / L / M / N / S / T / V / W / Y, 913F / I / K / M / N / S or 932C / D / E / G / H / K / L / M / N / P / Q / R / W / Y, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

3. The engineered acidic α-glucosidase according to claim 1 or 2, wherein the amino acid sequence of the engineered acidic α-glucosidase comprises residues 20 to 944 of SEQ ID NO. with even numbers in SEQ ID NO: 14 to 754, or comprises SEQ ID NO. with even numbers in SEQ ID NO: 14 to 754.

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

5. The engineered acidic α-glucosidase according to any one of claims 1 to 4, wherein, compared with a reference acidic α-glucosidase having a sequence of residues 20 to 944 corresponding to SEQ ID NO: 2 or 12 or a sequence corresponding to SEQ ID NO: 2 or 12, the engineered acidic α-glucosidase exhibits at least one improved property selected from: i) enhanced catalytic activity; ii) improved tolerance to pH 7; iii) improved tolerance to pH 4.4; iv) improved stability in lysosomes; v) Increased expression in cells; vi) Increased uptake into cells; vii) Increased enzymatic activity in cell lysates; viiii) Increased stability in plasma / serum; And ix) decreased immunogenicity; or a combination of any of i), ii), iii), iv), v), vi), vii), viiii), and ix).

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

7. The engineered acidic α-glucosidase according to claim 6, wherein the engineered acidic α-glucosidase exhibits the following: (a) a total immunogenicity score (TIS) reduction of more than 10 compared to the reference acidic α-glucosidase of SEQ ID NO: 2; (b) an immunogenicity hit count (IHC) reduction of more than 2 compared to the reference acidic α-glucosidase of SEQ ID NO: 2; (c) a total immunogenicity score (TIS) reduction of more than 10 compared to the reference acidic α-glucosidase of SEQ ID NO: 12; and / or (d) an immunogenicity hit count (IHC) reduction of more than 2 compared to the reference acidic α-glucosidase of SEQ ID NO:

12.

8. The engineered acidic α-glucosidase according to any one of claims 1 to 7, comprising the precursor peptide of the engineered acidic α-glucosidase.

9. The engineered acidic α-glucosidase according to claim 8, wherein the precursor peptide of the engineered acidic α-glucosidase comprises a eukaryotic or synthetic signal peptide sequence.

10. The engineered acidic α-glucosidase according to claim 9, wherein the signal peptide comprises a mouse or human signal peptide sequence.

11. The engineered acidic α-glucosidase according to any one of claims 1 to 7, comprising the peptide origin of the engineered acidic α-glucosidase.

12. The engineered acidic α-glucosidase according to any one of claims 1 to 11, wherein the engineered acidic α-glucosidase is purified.

13. A pharmaceutical composition comprising an engineered acidic α-glucosidase according to any one of claims 1 to 12.

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

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

16. A recombinant polynucleotide comprising a polynucleotide sequence encoding an engineered acidic α-glucosidase according to any one of claims 1 to 11.

17. The recombinant polynucleotide of claim 16, comprising a reference polynucleotide sequence comprising nucleotide residues 58 to 2832 of SEQ ID NO. corresponding to odd-numbered numbers in SEQ ID NO: 13 to 753 or a reference polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher sequence identity with a reference polynucleotide sequence corresponding to odd-numbered numbers in SEQ ID NO: 13 to 753, wherein the polynucleotide encodes an acidic α-glucosidase.

18. The recombinant polynucleotide of claim 16 or 17, comprising a polynucleotide sequence codon-optimized for expression of an engineered acidic α-glucosidase.

19. The recombinant polynucleotide of claim 16, comprising: a polynucleotide sequence comprising nucleotide residues 58 to 2832 of SEQ ID NO. of odd numbers from SEQ ID NO: 13 to 753; or a polynucleotide sequence comprising SEQ ID NO. of odd numbers from SEQ ID NO: 13 to 753.

20. The recombinant polynucleotide of claim 16, comprising: a polynucleotide sequence comprising nucleotide residues 58 to 2832 of SEQ ID NO: 13, 113, 125, 169, 249, 251, 393, 471, 487 or 505; or a polynucleotide sequence comprising SEQ ID NO: 13, 113, 125, 169, 249, 251, 393, 471, 487 or 505.

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

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

23. The expression vector of claim 22, wherein the control sequence comprises a promoter.

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

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

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

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

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

29. The host cell of claim 28, wherein the human cell is derived from a patient suffering from acid α-glucosidase activity deficiency.

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

31. The method of claim 30, further comprising the step of recovering the acidic α-glucosidase.

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

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

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

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

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

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

38. Use of the engineered acid α-glucosidase according to any one of claims 1 to 12 in the preparation of a medicament for treating a subject suffering from acid α-glucosidase deficiency.

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

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