CN118667797A - A recombinant acid alpha-glucosidase and its use - Google Patents

Abstract

The present invention discloses a recombinant acid α-glucosidase and its use. Based on the amino acid sequence of the parent Myozyme, ASP-GAA-3 is obtained by using an artificial signal peptide and removing the precursor peptide sequence, and the nucleic acid sequence of ASP-GAA-3 is inserted into the pEE17.4 vector, and a CHO stable cell line is constructed by electroporation, and the stable cell line monoclonal clone is expressed and purified to obtain high-purity rhGAA. This method can not only increase the expression amount of rhGAA, but also the precursor peptide does not need to be cut off during the post-expression processing, and the N-terminal sequence identical to the commercialized Myozyme can be directly obtained. The protein expressed by the stable cell line constructed using the ASP signal peptide and removing the precursor peptide sequence has an enzyme activity similar to that of Myozyme expressed by the NSP signal peptide.

Description

A recombinant acid alpha-glucosidase and its use

Technical Field

The invention belongs to the fields of molecular biology and bioengineering, and specifically relates to a recombinant acid alpha-glucosidase and a use thereof.

Background Art

Pompe disease, also known as glycogen storage disease type II, acid maltase deficiency or type II glycogenosis, is inherited in an autosomal recessive manner and is caused by a lack of acid α-glucosidase (GAA) in the lysosomes. GAA deficiency causes glycogen to be unable to be converted into glucose and utilized, resulting in a large amount of glycogen accumulating in tissue cells such as skeletal muscle, myocardium and smooth muscle, causing disease. Infantile patients develop the disease shortly after birth, with symptoms of severe hypotonia, weakness, liver enlargement, and heart enlargement. As the disease progresses, children gradually develop difficulty swallowing and a protruding and enlarged tongue. Most children die before the age of 2 due to respiratory or cardiac complications. Late-onset Pompe disease develops after infancy and is characterized by progressive muscle weakness, exercise intolerance, and gradual involvement of respiratory muscles leading to respiratory failure.

Prior to 2006, there were no products approved for the treatment of Pompe disease. Palliative care and supportive care strategies used by patients were largely ineffective in preventing disease progression. Early attempts to administer enzyme replacement therapy (ERT) to infants using enzyme preparations purified from Aspergillus niger or human placenta were unsuccessful, possibly due to dosing, disease stage, and lack of the correct posttranslational modifications required for muscle targeting. The first clinical study using GAA purified from transgenic rabbit milk showed that ERT could improve infant respiratory insufficiency and restore some muscle function. Subsequently, the safety and efficacy of recombinant human acid α-glucosidase (rhGAA) from two different Chinese hamster ovary (CHO) cell lines were evaluated in 18 infants with Pompe disease and the results showed that rhGAA was safe and effective for the treatment of infantile-onset Pompe disease.

The cDNA of human GAA encodes a protein of 952 amino acids with a predicted molecular weight of 105 kDa. The newly synthesized precursor protein has a signal peptide for co-translational transport into the lumen of the endoplasmic reticulum, where it undergoes N-glycosylation modification at seven glycosylation sites, thereby producing a glycosylated precursor with a size of 110 kDa. Further studies have shown that GAA is first synthesized as a 110 kDa glycosylated precursor containing an N-linked carbohydrate modified with a mannose 6-phosphate group. After being transported to the lysosome through the mannose 6-phosphate receptor, the 110 kDa precursor undergoes a series of complex proteolysis and N-glycan modification processes, producing two forms of the protein, 76 and 67 kDa.

Myozyme degrades glycogen by catalyzing the hydrolysis of α-1,4- and α-1,6-glycosidic bonds of glycogen. It is an enzyme replacement therapy first promoted by Genzyme and is suitable for patients with Pompe disease who are deficient in GAA. This drug is produced by recombinant DNA technology in CHO cell lines. The drug was launched in the EU and the United States in 2006, in Japan one year later, and approved in China in 2015. Myozyme is administered intravenously, and its recommended dose is 20 mg/kg body weight, once every 2 weeks. This dose is much higher than the recommended dose for the treatment of Gaucher disease and Fabry disease (1.5 and 1.0 mg/kg, respectively). At present, each 50 mg vial is priced at 5,480 yuan, which costs about 2 million yuan per patient per year. Therefore, the research and development of Pompe disease drugs and cost reduction are of great significance to patients with Pompe disease.

Based on the complex post-translational modification of GAA (mannose 6-phosphorylation), the first-generation recombinant enzyme (Myozyme) carefully utilized its own natural signal peptide (Natural signal peptide, NSP) and propeptide, and secreted rhGAA into the culture supernatant in the form of a 110kDa precursor using CHO cells through DNA recombination technology. In addition to the 110kDa precursor form, recombinant CHO cells also release other forms of proteolytically processed GAA into the culture medium through an undetermined mechanism. Due to its low expression level and unevenness, it poses many challenges to subsequent purification. Therefore, technicians in this field are committed to developing an rhGAA that can increase expression and increase uptake by cells and tissues that need it.

Summary of the invention

The object of the present invention is to provide a recombinant human acid alpha-glucosidase in view of the current status and existing deficiencies of the prior art. Based on the amino acid sequence of the parent Myozyme, by using an artificial signal peptide (ASP) and removing the propeptide sequence, a recombinant human acid alpha-glucosidase is obtained, and the nucleic acid sequence encoding the recombinant human acid alpha-glucosidase is inserted into the pEE17.4 vector, and a CHO stable cell line is constructed by electroporation, and the stable cell line monoclonal is expressed and purified to obtain high-purity rhGAA. This method can not only increase the expression amount of rhGAA, but also the post-expression processing process does not need to cut off the propeptide, and the N-terminal sequence identical to the commercialized Myozyme can be directly obtained. The protein expressed by the stable cell line constructed using the ASP signal peptide and removing the propeptide sequence has an enzyme activity similar to that of Myozyme expressed by the NSP signal peptide.

To achieve the above object, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides a recombinant human acid α-glucosidase, wherein the amino acid sequence of the recombinant human acid α-glucosidase is selected from any one of the following:

1) It comprises a signal peptide component, a precursor peptide sequence and a functional human acid α-glucosidase component fused to its N-terminal end, the amino acid sequence of the signal peptide component is shown in SEQ ID NO.2, the precursor peptide sequence and the functional human acid α-glucosidase component have at least 85%, more preferably at least 90%, even more preferably at least 92% identity with the sequence shown in positions 28-952 of the amino acid sequence of the parent Myozyme, in particular at least 95% identity, such as at least 98, 99 or 100% identity, and retains the function of the parent Myozyme, the amino acid sequence of the Myozyme is shown in SEQ ID NO.1;

2) It comprises a signal peptide component and a functional human acid α-glucosidase component fused to its N-terminal end, the amino acid sequence of the signal peptide component is shown in SEQ ID NO.2, the functional human acid α-glucosidase component is truncated by 56 consecutive amino acids at its N-terminal end compared with the amino acid sequence of the parent Myozyme, and the remaining sequence has at least 85%, more preferably at least 90%, even more preferably at least 92% identity with the sequence shown at positions 57-952, in particular at least 95% identity, such as at least 98, 99 or 100% identity, and retains the function of the parent Myozyme, the amino acid sequence of the Myozyme is shown in SEQ ID NO.1;

In a preferred embodiment, the amino acid sequence of the recombinant human acid α-glucosidase of the present invention is particularly shown in any one of SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5; the recombinant human acid α-glucosidase polypeptide is more particularly the amino acid sequence shown in SEQ ID NO.5.

In a second aspect, the present invention provides a nucleic acid molecule encoding the recombinant human acid α-glucosidase described in the first aspect;

In a preferred embodiment, the nucleotide sequence of the nucleic acid molecule has at least 85%, more preferably at least 90%, even more preferably at least 92% identity with the sequence shown in any one of SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8, in particular at least 95% identity, for example at least 98, 99 or 100% identity, and retains the function of the sequence shown in any one of SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8; the nucleic acid molecule is more particularly the nucleic acid sequence shown in SEQ ID NO.8.

In a third aspect, the present invention provides a nucleic acid construct comprising the nucleic acid molecule of the second aspect of the present invention operably linked to one or more regulatory sequences, such as a promoter, a Kozak sequence;

In a specific embodiment, the promoter is preferably selected from the murine cytomegalovirus (mCMV) promoter, and the Kozak sequence is GCCACC; in a specific embodiment of the nucleic acid construct of the present invention, the nucleic acid construct preferably comprises, in the following order: mCMV, 5' untranslated region, intron, kozak sequence, and the nucleic acid molecule described in the second aspect of the present invention.

In a fourth aspect, the present invention relates to a vector comprising the nucleic acid molecule described in the second aspect or the nucleic acid construct described in the third aspect;

In a specific embodiment, the nucleic acid construct comprises the nucleotide sequence of SEQ ID NO.9.

In a fifth aspect, the present invention provides a cell transformed with the nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect, or the vector of the fourth aspect;

More specifically, the cells are CHOK1SV GS-KO cells.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising the nucleic acid molecule described in the second aspect, the nucleic acid construct described in the third aspect, the vector described in the fourth aspect, or the cell described in the fifth aspect in a pharmaceutically acceptable carrier.

In a seventh aspect, the present invention provides use of the recombinant human acid α-glucosidase described in the first aspect, the nucleic acid component described in the second aspect, the nucleic acid construct described in the third aspect, the vector described in the fourth aspect, or the cell described in the fifth aspect in the preparation of a drug for treating glycogen storage disease;

In a preferred embodiment, the glycogen storage disease is glycogen storage disease type II.

Beneficial effects of the present invention:

Compared with the natural signal peptide (NSP), the present invention uses an artificial signal peptide (ASP) to express rhGAA, and the expression amount is similar. Experiments have found that after using the artificial signal peptide and removing the propeptide sequence (ASP-GAA-2, ASP-GAA-3), the expression amount is improved. It is predicted that the expression of GAA by the ASP signal peptide and the removal of the propeptide sequence is a good choice, which can produce unexpected results, significantly improve the expression amount, and have better stability. This method can not only increase the expression amount of GAA, but also the post-expression processing process does not need to cut off the propeptide, and the N-terminal sequence identical to the commercialized Myozyme can be directly obtained. The protein expressed by the stable cell line constructed by using the ASP signal peptide and removing the propeptide sequence has an enzyme activity similar to that of Myozyme expressed by the NSP signal peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Electrophoresis of the supernatant of Expi293 cells expressing rhGAA;

Figure 2. pEE17.4 vector map;

Figure 3. Electrophoresis of the supernatant of CHOK1SV GS-KO cells expressing rhGAA;

Figure 4. Product 4-MU standard curve;

Figure 5. Enzyme activity test results of rhGAA and commercial Myozyme;

DETAILED DESCRIPTION

The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

Definition and explanation:

As used herein, the term "GAA" or "GAA polypeptide" encompasses mature (-76 or -67 kDa) and precursor (e.g., -110 kDa) GAA, in particular the precursor form, as well as GAA proteins or fragments thereof modified or mutated by insertion, deletion and/or substitution, which are functional derivatives of GAA, i.e., they retain the biological function of GAA (i.e., have at least one biological activity of the native GAA protein as defined above, e.g., can hydrolyze glycogen), and GAA variants (e.g., GAA II described by Kunita et al., (1997) Biochemica et Biophysica Acta 1362:269; GAA II described by Hirschhorn, R. and Reuser, A.J. (2001) in The Metabolic and Molecular Basis for Inherited Diseases. Disease) (Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D., eds.), pp. 3389-3419, McGraw-Hill, New York, GAA polymorphisms and SNPs described, see pp. 3403-3405). Any GAA coding sequence known in the art can be used, for example, see SEQ ID NO: 1; GenBank Accession No. NM_00152, Hoefsloot et al., (1988) EMBO J. 7: 1697 and Van Hove et al., (1996) Proc. Natl. Acad. Sci. USA 93: 65 (human), GenBank Accession No. NM_008064 (mouse), and Kunita et al., (1997) Biochemica et Biophysica Acta 1362: 269 (quail).

In the context of the present invention, a "precursor form of GAA" is a form of a GAA polypeptide that includes its native signal peptide. For example, the sequence of SEQ ID NO: 1 is a precursor form of recombinant human acid α-glucosidase. In SEQ ID NO: 1, amino acid residues 1-27 correspond to the signal peptide of the hGAA polypeptide.

The term "rhGAA" is intended to refer to recombinant human acid alpha-glucosidase and is used to distinguish endogenous GAA from synthetic or recombinantly produced GAA (e.g., GAA produced by CHO cells transformed with DNA encoding GAA). The term "rhGAA" encompasses a population of individual rhGAA molecules. Provided herein are properties of a population of rhGAA molecules.

The term "commercial rhGAA products" is intended to refer to products containing alglucosidase alfa, such as or

In the context of the present invention, the recombinant human acid α-glucosidase of the present invention is derived from a parent GAA polypeptide. According to the present invention, a "parent GAA polypeptide" is an "amino acid sequence of a parent Myozyme", for example, with reference to a wild-type human GAA polypeptide, a complete wild-type GAA polypeptide (i.e., a precursor form of GAA) is shown in SEQ ID NO: 1, and has a signal peptide (corresponding to amino acids 1-27 of SEQ ID NO: 1). In this example, amino acids 57-952 corresponding to SEQ ID NO: 1 are referred to as functional human acid α-glucosidase components of the recombinant human acid α-glucosidase.

In certain embodiments, for modified nucleic acids encoding GAA, the GAA protein retains at least a portion of the function or activity of the wild-type GAA protein. The function or activity of the GAA protein includes acid alpha-glucosidase activity, a lysosomal hydrolase that degrades glycogen, maltose, and isomaltose. Thus, modified nucleic acids encoding GAA include modified forms as long as the encoded GAA retains a degree or aspects of the lysosomal hydrolase activity of GAA.

The terms "identity", "homology" and grammatical variations thereof refer to two or more referenced entities being identical when the sequences are "aligned". Thus, for example, when two nucleic acids are identical, they have identical sequences at least over a reference region or portion. The identity may be within a defined region (region or domain) of the sequence.

A "region" or "region" of identity refers to the identical portions of two or more referenced entities. Thus, when two protein or nucleic acid sequences are identical within one or more sequence regions or regions, they share identity within that region. An "aligned" sequence refers to a plurality of protein (amino acid) or nucleic acid sequences, typically including corrections for missing or extra bases or amino acids (gaps) compared to a reference sequence.

The degree of identity (homology) or "percent identity" between two sequences can be determined using computer programs and/or mathematical algorithms. For purposes of the present invention, the comparison of nucleotide sequences is performed using the GCG Wisconsin Package 9.1 version available from the Genetics Computer Group in Madison, Wisconsin. For convenience, the default parameters specified by the program (gap creation penalty = 12, gap extension penalty = 4) are intended to be used for comparing sequence identities. Alternatively, the Blastn2.0 program (available on the World Wide Web at ncbi.nlm.nih.gov/blast/) using gap alignments using default parameters provided by the National center for Biotechnology Information can be used to determine identity and similarity levels between nucleotide sequences and amino acid sequences. For polypeptide sequence comparison, the BLASTP algorithm is usually used in combination with a scoring matrix (e.g., PAM100, PAM 250, BLOSUM 62, or BLOSUM 50). FASTA (e.g., FASTA2 and FASTA3) and SSEARCH sequence comparison programs are also used to quantify the degree of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988); Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J. Mol. Biol. 147:195 (1981)). Programs for quantitative protein structural similarity using Delaunay-based topological maps have also been developed (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

The term "vector" refers to a small carrier nucleic acid molecule, plasmid, virus (e.g., AAV vector) or other vehicle that can be manipulated by insertion or incorporation of nucleic acid. Such vectors can be used for genetic manipulation (i.e., "cloning vectors"), to introduce/transfer polynucleotides into cells, and to transcribe or translate the inserted polynucleotides in cells. "Expression vectors" are specialized vectors that contain a gene or nucleic acid sequence with the necessary regulatory regions required for expression in a host cell.

The vector nucleic acid sequence generally comprises at least an origin of replication for propagation in the cell and optionally other elements, such as heterologous polynucleotide sequences, expression control elements (e.g., promoters, enhancers), introns, inverted terminal repeats (ITRs), selectable markers (e.g., antibiotic resistance), polyadenylation signals.

"Transgene" is used herein to conveniently refer to a heterologous nucleic acid that is intended to be or has been introduced into a cell or organism. A transgene includes any heterologous nucleic acid, such as a modified nucleic acid encoding GAA.

A "transduced cell" is a cell into which a transgene has been introduced. Thus, a "transduced" cell (e.g., in a mammal, such as a cell, tissue or organ cell) refers to a genetic change in the cell after, for example, a nucleic acid (e.g., a transgene) has been incorporated into the cell. Thus, a "transduced" cell is a cell or its progeny into which an exogenous nucleic acid has been introduced. The cell can reproduce and express the introduced protein. For purposes and methods of gene therapy, the transduced cell can be in a subject.

Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

Example 1: Synthesis of 4 different plasmids for expression of rhGAA in expi293 cells and quantification of expression in supernatant

NSP-GAA-PZD, ASP-GAA-1-PZD, ASP-GAA-2-PZD, and ASP-GAA-3-PZD were synthesized at Jinweizhi. The amino acid sequences of NSP-GAA, ASP-GAA-1, ASP-GAA-2, and ASP-GAA-3 are shown in SEQ ID NO: 1, 3, 4, and 5. DH10B was transformed separately, cultured overnight at 37°C, and 1 single clone was picked into 50mL LB medium, cultured overnight at 37°C, and plasmids were extracted using the Qiagen plasmid extraction kit. The plasmid was premixed with PEI at a ratio of 1:5 and transfected into expi293 cells. After 6 days, the supernatant was collected and the cell debris was removed. 20μL of supernatant was taken for each expression, 5μL of protein loading buffer was added, the sample was boiled at 95°C for 5 minutes, and 12% SDS-protein electrophoresis was run. The results are shown in Figure 1.

Octet RED96e was used to quantify the supernatant. Two 96-well plates were prepared, one as a sample plate and the other as a pre-wet plate. 200 μL of dilution buffer (PBS + 0.02% Tween 20 + 0.1% BSA) was added to each well of the pre-wet plate and placed in the blue bottom plate. The AMC sensor was placed on the green plate (sensor plate) at the corresponding position of the pre-wet well. The commercial standard was diluted to 100 μg/mL, 50 μg/mL, 25 μg/mL, 12.5 μg/mL, 6.25 μg/mL, and 3.125 μg/mL using dilution buffer, and 200 μL/well was added to the sample plate. After the cell supernatant was diluted 4 times with dilution buffer, 200 μL was taken and added to the sample plate. The Advanced Quantitation program was selected for expression detection, and the data were processed using Octet RED96e (ForteBio) analysis software Data Analysis11 software. The results are shown in Table 1. The gel image and quantitative results showed that the expression level increased by 3 times after using artificial signal peptide and removing the propeptide sequence.

Table 1. Quantitative results of the supernatant of Expi293 cells expressing rhGAA

Example 2. Construction of rhGAA stable cell line and protein preparation

Add Kozak sequence before the start codon of ASP-GAA-3, add HindIII restriction site at the N-terminus, add stop codon and EcoRI restriction site at the C-terminus, and connect to pEE17.4 (vector map shown in Figure 2) to construct PEE17.4/ASP-GAA-3 plasmid for sequencing. Extract the constructed PEE17.4/ASP-GAA-3 plasmid, operate according to the operating procedures provided by Lonza, and electrotransfer into CHOK1SV GS-KO cells to construct a stable cell line. Select a highly expressed monoclonal cell line and express it according to the culture method provided by Lonza. Harvest the cell supernatant after 12 days of expression. Take 20μL of supernatant, add 5μL protein loading buffer, and boil the sample at 95℃ for 5 minutes. Run 12% SDS-protein electrophoresis, the results are shown in Figure 3. At the same time, the cell supernatant was quantified, and the results are shown in Table 2. The gel images and quantitative results showed that the expression level of rhGAA could reach above 1.8 g/L by screening stable cell lines.

Table 2. Quantitative results of supernatant of CHOK1SV GS-KO cells expressing rhGAA

name GAA concentration 1889 μg/mL

The harvested cell solution was placed in a centrifuge and centrifuged at 10,000 g for 15 min. The supernatant was filtered using a 0.2 μm sterilizing filter. The filtered sample was used for subsequent chromatography experiments. 1) Equilibration: 5CV, equilibrium buffer: 20mM PB, 300mM NaCl, pH6.5; 2) Loading: harvest supernatant; 3) Wash1: buffer: 20mM phosphate buffer (PB), 300mM NaCl, 15% propylene glycol, pH6.5; 4) Wash2: 20mM PB, 300mM NaCl, pH6.5; 5) Elution: 20mM PB, 1MGlycine, pH6.3; 6) CIP: 0.5M NaOH, use ultrafiltration tube or desalting column to change salt to preparation buffer, and store in -80℃ refrigerator (Preparation buffer: 20mM PB, 20mg/ml mannitol, 0.5% polysorbate 80, pH6.0).

Example 3. rhGAA and commercial Myozyme protease activity assay

Preparation of standard curve: The product (4-Methylumbelliferone, 4-MU) was diluted with DMSO to 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0mM; the above products were diluted 100 times with Assay Buffer (0.2M sodium acetate, 0.02% lauryl alcohol polyoxyethylene ether); then the above products were diluted 10 times with 0.2M sodium bicarbonate solution, and the final concentrations were 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 and 0μM. 100μL of the diluted product was added to a 96-well fluorescence detection plate, and the parameters of the microplate reader (fluorescence intensity: excitation 360/40nm, emission 460/40nm) were set to read the results, as shown in Figure 4.

Enzyme activity reaction: Use Assay Buffer to dilute the substrate (4-Methylumbelliferyl-α-D-Glucopyranoside, 4-MUDG) to 4mM, add 10μL/well to a 96-well PCR plate, and place on ice for use; dilute the commercial Myozyme and the test sample rhGAA prepared in Example 2 to 25μg/mL with Assay Buffer, take 10μL of the diluted commercial Myozyme and the test sample rhGAA prepared in Example 2 and add them to the 96-well PCR plate containing 10μL of substrate, then place it in a PCR instrument, and start the program of the PCR instrument preset program (37℃, 6min; 4℃, ∞; no need for hot cover) to perform enzymatic reaction. After the reaction is completed, take out the 96-well PCR plate from the PCR instrument and place it on ice, add 180μL of 0.2M sodium bicarbonate solution to mix well, and terminate the reaction. 100 μL of the above reaction solution was added to a 96-well fluorescence detection plate, and the parameters of the microplate reader were set (fluorescence intensity: excitation 360/40 nm, emission 460/40 nm), and the reading was performed. The results are shown in Figure 5. The protein expressed by the stable cell line constructed using the ASP signal peptide and removing the precursor peptide sequence has an enzyme activity similar to that of Myozyme expressed by the NSP signal peptide.

The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. A recombinant human acid alpha-glucosidase, characterized in that the amino acid sequence of the recombinant human acid alpha-glucosidase is selected from any of the following:

1) Comprising a signal peptide moiety fused to its N-terminal end, a precursor peptide sequence and a functional human acidic α -glucosidase moiety, the amino acid sequence of which is shown in SEQ ID No.2, the precursor peptide sequence and the functional human acidic α -glucosidase moiety having at least 85%, more preferably at least 90%, even more preferably at least 92% identity, in particular at least 95% identity, for example at least 98, 99 or 100% identity, to the sequence shown at positions 28-952 of the amino acid sequence of the parent Myozyme, the amino acid sequence of which is shown in SEQ ID No.1, and retaining the function of the parent Myozyme;

2) Comprising a signal peptide moiety fused to its N-terminal end, the amino acid sequence of which is shown as SEQ ID No.2, and a functional human acid alpha-glucosidase moiety truncated at its N-terminal end by 56 consecutive amino acids compared to the amino acid sequence of the parent Myozyme, the remaining sequence having at least 85%, more preferably at least 90%, even more preferably at least 92% identity, in particular at least 95% identity, for example at least 98, 99 or 100% identity, to the sequence shown at positions 57-952, and retaining the function of the parent Myozyme, the amino acid sequence of which is shown as SEQ ID No. 1.

2. The recombinant human acid alpha-glucosidase of claim 1, wherein the amino acid sequence of the recombinant human acid alpha-glucosidase is set forth in any of SEQ ID No.3, SEQ ID No.4, or SEQ ID No. 5; preferably, the recombinant human acid alpha-glucosidase polypeptide is the amino acid sequence shown in SEQ ID NO. 5.

3. A nucleic acid molecule encoding the recombinant human acid α -glucosidase of claim 1 or 2.

4. A nucleic acid molecule according to claim 3, characterized in that the nucleotide sequence of the nucleic acid molecule has at least 85%, preferably at least 90%, more preferably at least 92% identity, in particular at least 95% identity, such as at least 98, 99 or 100% identity, with the sequence shown in any one of SEQ ID No.6, SEQ ID No.7 or SEQ ID No.8 and retains the function of the sequence shown in any one of SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8; the nucleic acid molecule is more particularly the nucleic acid sequence shown in SEQ ID NO. 8.

5. A nucleic acid construct comprising a nucleic acid molecule according to claim 3 or 4 operably linked to one or more regulatory sequences, such as a promoter, kozak sequence;

Preferably, the promoter is selected from murine cytomegalovirus (mCMV) promoter, the Kozak sequence is GCCACC; in a particular embodiment of the nucleic acid construct of the invention, the nucleic acid construct preferably comprises, in the following order: mCMV, 5' untranslated region, intron, kozak sequence, nucleic acid molecule according to claim 3 or 4.

6. A vector comprising the nucleic acid molecule of claim 3 or 4 or the nucleic acid construct of claim 5;

In a specific embodiment, the vector comprises the nucleotide sequence of SEQ ID No. 9.

7. A cell transformed with the nucleic acid molecule of claim 3 or4 or the nucleic acid construct of claim 5 or the vector of claim 6 into a recipient cell;

in a specific embodiment, the recipient cell is a CHOK1SV GS-KO cell.

8. A pharmaceutical composition comprising the nucleic acid molecule of claim 3 or 4 or the nucleic acid construct of claim 5 or the vector of claim 6 or the cell of claim 7 in a pharmaceutically acceptable carrier.

9. Use of the recombinant human acid α -glucosidase of claim 1 or 2, the nucleic acid molecule of claim 3 or 4, the nucleic acid construct of claim 5, the vector of claim 6, or the cell of claim 7 in the preparation of a medicament for treating a glycogen storage disease.

10. The use according to claim 9, wherein the glycogen storage disease is glycogen storage disease type II.